Mechanisms of Podocyte Injury in Diabetes: Role of Cytochrome P450 and NADPH Oxidases

OBJECTIVE

We investigated the role of cytochrome P450 of the 4A family (CYP4A), its metabolites, and NADPH oxidases both in reactive oxygen species (ROS) production and apoptosis of podocytes exposed to high glucose and in OVE26 mice, a model of type 1 diabetes.

RESEARCH DESIGN AND METHODS

Apoptosis, albuminuria, ROS generation, NADPH superoxide generation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo.

RESULTS

Exposure of mouse podocytes to high glucose resulted in apoptosis, with approximately one-third of the cells being apoptotic by 72 h. High-glucose treatment increased ROS generation and was associated with sequential upregulation of CYP4A and an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) and Nox oxidases. This is consistent with the observation of delayed induction of NADPH oxidase activity by high glucose. The effects of high glucose on NADPH oxidase activity, Nox proteins and mRNA expression, and apoptosis were blocked by N-hydroxy-N′-(4-butyl-2-methylphenol) formamidine (HET0016), an inhibitor of CYP4A, and were mimicked by 20-HETE. CYP4A and Nox oxidase expression was upregulated in glomeruli of type 1 diabetic OVE26 mice. Treatment of OVE26 mice with HET0016 decreased NADPH oxidase activity and Nox1 and Nox4 protein expression and ameliorated apoptosis and albuminuria.

CONCLUSIONS

Generation of ROS by CYP4A monooxygenases, 20-HETE, and Nox oxidases is involved in podocyte apoptosis in vitro and in vivo. Inhibition of selected cytochrome P450 isoforms prevented podocyte apoptosis and reduced proteinuria in diabetes.Diabetic nephropathy in humans is characterized by increased urinary albumin excretion (microalbuminuria), which often progresses to proteinuria, one of the most important prognostic risk factors for kidney disease progression (1). Glomerular visceral epithelial cells, or podocytes, play a critical role in maintaining the structure and function of the glomerular filtration barrier. Careful morphometric analyses of renal biopsy in subjects with type 1 and type 2 diabetes (2–4) demonstrate that the density of podocytes is reduced not only in individuals with diabetic nephropathy, but also in patients with short duration of diabetes before the onset of microalbuminuria (4,5). Studies in experimental models of type 1 and type 2 diabetes have also documented that podocyte depletion represents one of the earliest cellular lesions affecting the diabetic kidney (6,7). Among various morphologic characteristics, the decreased number of podocytes in glomeruli is the strongest predictor of progression of diabetic nephropathy, where fewer cells predict more rapid progression (3,4). Although these observations identify podocyte depletion as one of the earliest cellular features of diabetic kidney disease, the mechanisms that underlie the loss of podocytes in diabetic nephropathy remain poorly understood.High glucose induces apoptosis (8), and there is evidence that podocyte apoptosis contributes to reduced podocyte number (9). High glucose, transforming growth factor-β (TGF-β), and angiotensin II (ANGII) induce apoptosis of cultured podocytes (9–12). ANGII appears to induce apoptosis in cultured rat glomerular epithelial cells at least partially via TGF-β because its apoptotic effect is attenuated by an anti–TGF-β antibody (12). There is also evidence that reactive oxygen species (ROS) contribute to podocyte apoptosis and depletion in cells exposed to high glucose and in experimental diabetic nephropathy (7). However, the sources of ROS and the kinetics of their generation have not been well characterized. We and others (13–15) have recently identified NADPH oxidases as major sources of ROS in kidney cortex and glomeruli of rats with type 1 diabetes. Six homologs of the cytochrome subunit of the phagocyte NADPH oxidase (Nox2/gp91^phox) have been cloned (16). At least three different Nox isoforms are expressed in the kidney cortex: Nox1, Nox2, and Nox4 (16). Cytochromes P450 (CYP450s) are significant sources of ROS in many tissues (17,18). CYP450 metabolizes arachidonic acid into hydroxyeicosatetraenoic acids (20-HETEs) and EETs (epoxyeicosatrienoic acids). 20-HETE, the ω-hydroxylation product of arachidonic acid, is one of the major CYP eicosanoids produced in the kidney cortex (19–21). The predominant CYP450 in the kidney cortex that synthesizes 20-HETE is cytochrome P450 of the 4A family (CYP4A) (19–21). 20-HETE has multiple and opposing functions depending on the site of production and target cells/tissues (19,22–24).In this study, we demonstrate that high glucose induces ROS production and apoptosis in cultured mouse podocytes through the upregulation of CYP4A with increased production of 20-HETE and upregulation of NADPH oxidases. Inhibition of 20-HETE production prevented podocyte apoptosis in vitro and decreased oxidative stress, podocyte apoptosis, and proteinuria in an in vivo model of type 1 diabetes.     

Effect of the Monocyte Chemoattractant Protein-1/CC Chemokine Receptor 2 System on Nephrin Expression in Streptozotocin-Treated Mice and Human Cultured Podocytes

OBJECTIVE

Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes.

RESEARCH DESIGN AND METHODS

Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting.

RESULTS

In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin.

CONCLUSIONS

These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes.Diabetic nephropathy is characterized by increased glomerular permeability to proteins (1). Recently, much attention has been paid to the role of podocyte injury in glomerular diseases, including diabetic nephropathy (2,3), but the precise molecular mechanisms underlying the development of diabetic proteinuria remain unclear.The slit diaphragm, a junction connecting foot processes of neighboring podocytes, represents the major restriction site to protein filtration (4). Mutations of the gene encoding for nephrin, a key component of the slit diaphragm, are responsible for the congenital nephrotic syndrome of the Finnish type (5). Furthermore, a link between a reduction in nephrin expression and proteinuria has been also reported in acquired proteinuric conditions, including diabetic nephropathy (6–8), and studies in patients with incipient diabetic nephropathy have demonstrated that nephrin downregulation occurs in an early stage of the disease (9).A number of factors, including high glucose, advanced glycation end products, and hypertension play a role in the pathogenesis of diabetic nephropathy (10). In addition, monocyte chemoattractant protein-1 (MCP-1), a potent mononuclear cell chemoattractant, is overexpressed within the glomeruli in experimental diabetes (11,12) and has been recently implicated in both functional and structural abnormalities of the diabetic kidney (13).MCP-1 binds to the cognate CC chemokine receptor 2 (CCR2), which is predominantly expressed on monocytes (14), and MCP-1–driven monocyte accrual is considered the predominant mechanism whereby MCP-1 contributes to the glomerular damage. However, the CCR2 receptor has also been shown both in vitro (15,16) and in vivo (17–19) in other cell types besides monocytes, and we have recently demonstrated that both mesangial cells and glomerular podocytes express a functionally active CCR2 receptor (20–22).To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin, expression in podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and/or expression of nephrin in experimental diabetes.     

Inhibition of Podocyte FAK Protects against Proteinuria and Foot Process Effacement

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays a critical role in cell motility. Movement and retraction of podocyte foot processes, which accompany podocyte injury, suggest focal adhesion disassembly. To understand better the mechanisms by which podocyte foot process effacement leads to proteinuria and kidney failure, we studied the function of FAK in podocytes. In murine models, glomerular injury led to activation of podocyte FAK, followed by proteinuria and foot process effacement. Both podocyte-specific deletion of FAK and pharmacologic inactivation of FAK abrogated the proteinuria and foot process effacement induced by glomerular injury. In vitro, podocytes isolated from conditional FAK knockout mice demonstrated reduced spreading and migration; pharmacologic inactivation of FAK had similar effects on wild-type podocytes. In conclusion, FAK activation regulates podocyte foot process effacement, suggesting that pharmacologic inhibition of this signaling cascade may have therapeutic potential in the setting of glomerular injury.The glomerulus forms the filtration barrier of the kidney and is composed of a fenestrated endothelium, glomerular basement membrane (GBM), and the podocytes that interdigitate to form slit diaphragms.^1,2 When the podocytes are damaged, foot process fusion occurs. This process involves the rearrangement of the actin cytoskeleton and retraction of the foot processes toward the cell body, allowing mechanical forces and signaling events to be transmitted into the cell. Since the identification that mutations of the podocyte slit diaphragm specific NPHS1 gene cause congenital nephrotic syndrome,^3–5 podocytes have been recognized as critical regulators of glomerular injury. Other podocyte slit diaphragm proteins such as podocin, synaptopodin, and CD2AP have generated further interest in the regulation of the kidney filtration barrier^6–8; however, little is still known about cell–matrix interactions in podocytes. Mice lacking the focal adhesion protein integrin-linked kinase (ILK), specifically in the podocytes, also develop proteinuria, resulting in renal failure and death.⁹ Moreover, mice lacking α3β1 integrin have demonstrated inability to form mature foot processes.¹⁰ These cell–matrix interactions, which seem important in podocyte development, may also play a critical role after podocyte injury, because the process of podocyte effacement requires cell process retraction and movement, processes that suggest focal adhesion disassembly.Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase, in which integrin- or growth factor–induced autophosphorylation at tyrosine 397 results in activation of critical signaling pathways required for focal adhesion turnover.^11–16 It has been demonstrated that cell spreading and migration are significantly diminished in cells lacking FAK.¹⁷ This inhibition in motility has brought excitement in cancer therapeutics, resulting in the development and use of FAK inhibitors.^18–21 In a recent study, inhibition of urokinase plasminogen activator (uPAR), a glycosylphosphatidylinositol-anchored protein that is important for cell invasion and metastasis, has been demonstrated to reduce proteinuria and podocyte effacement significantly, suggesting that this dynamic podocyte cell movement may mimic the molecular signaling events observed in cancer cell invasion.²²In this study, we demonstrated that after podocyte injury in vivo and in vitro, FAK activation was significantly increased in wild-type (WT) mice, prompting us to address whether inhibition or loss of FAK activation would reduce podocyte cell motility by inhibiting focal adhesion turnover, thereby preventing proteinuria and effacement. Because complete FAK gene deletion results in lethality at embryonic day 8.5, a time point before glomerular development has been initiated, the ability to study this protein''s role in podocyte development as well as repair after injury has been limited.¹⁷ Hence, selective loss of FAK expression in the podocytes of the kidney was achieved using a Cre-loxP approach.^23,24 These mice were born without evidence of podocyte/glomerular developmental defects but were resistant to the foot process fusion and subsequent proteinuria that typically accompany LPS and rabbit anti-mouse GBM-induced podocyte damage. We postulate this inhibition of foot process effacement is due to diminished podocyte spreading and motility, supported by our in vitro data. In addition, pharmacologic treatment of WT mice using the FAK inhibitor TAE-226 significantly reduced proteinuria and podocyte effacement, raising the possibility for therapeutic use in glomerular diseases.     

Adiponectin Promotes Functional Recovery after Podocyte Ablation

Low levels of the adipocyte-secreted protein adiponectin correlate with albuminuria in both mice and humans, but whether adiponectin has a causative role in modulating renal disease is unknown. Here, we first generated a mouse model that allows induction of caspase-8–mediated apoptosis specifically in podocytes upon injection of a construct-specific agent. These POD-ATTAC mice exhibited significant kidney damage, mimicking aspects of human renal disease, such as foot process effacement, mesangial expansion, and glomerulosclerosis. After the initial induction, both podocytes and filtration function recovered. Next, we crossed POD-ATTAC mice with mice lacking or overexpressing adiponectin. POD-ATTAC mice lacking adiponectin developed irreversible albuminuria and renal failure; conversely, POD-ATTAC mice overexpressing adiponectin recovered more rapidly and exhibited less interstitial fibrosis. In conclusion, these results suggest that adiponectin is a renoprotective protein after podocyte injury. Furthermore, the POD-ATTAC mouse provides a platform for further studies, allowing precise timing of podocyte injury and regeneration.CKD, either as a primary renal abnormality or resulting from systemic conditions, such as diabetes or hypertension, afflicts an increasing number of Americans each year.¹ Reduced GFR, which is a hallmark of CKD, may be present in upwards of 20 million patients in the United States.^1,2 Within the glomerulus, podocytes form intricate foot processes, whose specialized contacts wrap glomerular capillaries forming the slit diaphragms that restrict large proteins from entering the urine. Selective injury to podocytes can result in primary diseases, such as FSGS with podocyte loss.^3,4 Podocyte loss also occurs in systemic diseases, such as early diabetic nephropathy,⁵ obesity, and the metabolic syndrome,⁶ resulting in a sclerosing lesion and potential progression to CKD.⁷ Although the subsequent progression of these nephropathies of diverse causes may follow different paths, the loss of or injury to podocytes has become increasingly appreciated as a common starting point.Albuminuria and diminished GFR are hallmarks of failing glomerular function leading to progressive sclerosis and eventual renal failure. Several rodent models have been developed to address specific aspects of this progression. Rodent remnant models, such as uninephrectomies and 5/6 nephrectomies, and various chemical injury models have multiple effects on different nephron structures.^8,9 Genetic manipulations of podocytes in mice and rats have demonstrated the importance of specific surface and structural proteins on podocyte function or rendered the models’ podocytes more susceptible to injury or cytotoxic ablation.^4,10–12 Correlating many of these models to initial human podocyte loss, however, has not always been possible because of design limitations or non-physiologic pathologic progression. Here we present a novel model of podocyte apoptosis that relies on the endogenous caspase-8 apoptotic pathway to permit inducible, selective ablation of podocytes from the glomerulus.We generated a mouse using the human podocin promoter (Nphs2) to drive expression of a mutant FKBP fusion protein that, once dimerized, induces physiologic, caspase-8–mediated apoptosis in podocytes. Dimerization and caspase-8 activation occurs upon injection of a low-molecular-weight, construct-specific agent (AP20187) that uniquely binds only to the FKBP mutant protein.¹³ This ATTAC (apoptosis through targeted activation of caspase-8) gene construct expression strategy has been successfully used in adipocytes,¹³ pancreatic β cells,¹⁴ and cardiac myocytes,¹⁵ with each model demonstrating dose-dependent apoptosis with restored tissue function over time. The endogenous nature of ATTAC-induced ablation is also not inflammatory.¹⁶ Our new podocyte-specific POD-ATTAC mouse thus allows us to mimic the first step in many forms of human glomerulosclerosis—loss of podocytes—and study the resultant nephropathic progression, potential recovery, and long-term sequelae.Adiponectin is an adipocyte-secreted protein that has positive effects on insulin sensitivity and cardiovascular disease.^17,18 Unlike other adipokines that increase with weight gain, serum adiponectin is inversely correlated with obesity. Low adiponectin has been correlated with albuminuria in both mice and humans, and obesity (and, therefore, low adiponectin) may be causative in some patients with FSGS.^6,7,19,20 Adiponectin knockout (KO) mouse models demonstrate increased podocyte injury and albuminuria, with adiponectin therapies potentially restoring some renal function.^21,22 Interestingly, the relationship is inversed in human CKD: Adiponectin levels are greatly elevated in both children and adults with CKD, with serum levels directly correlated to proteinuria.^20,23 Whether adiponectin merely correlates with the disease states associated with CKD or plays a direct role in renal function remains to be clarified. We have recently demonstrated that transgenic mice overexpressing adiponectin (we call them “adipo Tg” mice) are protected from caspase-8–mediated apoptosis and loss of organ function.¹⁵ These mice and our adiponectin KO mice (all crossed with our POD-ATTAC mice) furnish us with a valuable model to examine the role of adiponectin levels in the context of specific, inducible podocyte loss.The POD-ATTAC (POD) mouse exhibits inducible and titratable podocyte reduction and dose-dependent changes in GFR and albuminuria. Minimal nephropathic changes occur at low induction, but glomerular histologic changes approximating human FSGS are seen with higher dimerizer dose. At intermediate dimerizer doses, POD mice recover podocyte density, reverse foot process effacement, and restore filtration function, but at higher doses they develop interstitial fibrosis and fail to recover. Adiponectin KO POD mice are in every aspect worse off than wild-type adiponectin POD mice after equivalent podocyte ablation. Adipo Tg-POD mice lose numbers of podocytes similar to those lost by wild-type mice but are protected from fibrosis and are better able to restore function. With the ability to present a range of nephropathic features dependent on the degree of podocyte loss, the POD-ATTAC mouse provides a tightly controlled novel murine platform for studying the progression of CKD with the potential of recovery.     

Origin of Parietal Podocytes in Atubular Glomeruli Mapped by Lineage Tracing

Parietal podocytes are fully differentiated podocytes lining Bowman’s capsule where normally only parietal epithelial cells (PECs) are found. Parietal podocytes form throughout life and are regularly observed in human biopsies, particularly in atubular glomeruli of diseased kidneys; however, the origin of parietal podocytes is unresolved. To assess the capacity of PECs to transdifferentiate into parietal podocytes, we developed and characterized a novel method for creating atubular glomeruli by electrocoagulation of the renal cortex in mice. Electrocoagulation produced multiple atubular glomeruli containing PECs as well as parietal podocytes that projected from the vascular pole and lined Bowman’s capsule. Notably, induction of cell death was evident in some PECs. In contrast, Bowman’s capsules of control animals and normal glomeruli of electrocoagulated kidneys rarely contained podocytes. PECs and podocytes were traced by inducible and irreversible genetic tagging using triple transgenic mice (PEC- or Pod-rtTA/LC1/R26R). Examination of serial cryosections indicated that visceral podocytes migrated onto Bowman’s capsule via the vascular stalk; direct transdifferentiation from PECs to podocytes was not observed. Similar results were obtained in a unilateral ureter obstruction model and in human diseased kidney biopsies, in which overlap of PEC- or podocyte-specific antibody staining indicative of gradual differentiation did not occur. These results suggest that induction of atubular glomeruli leads to ablation of PECs and subsequent migration of visceral podocytes onto Bowman’s capsule, rather than transdifferentiation from PECs to parietal podocytes.Multiple studies have reported the presence of podocytes on Bowman’s capsule in normal¹ and diseased kidneys, close to the vascular pole in particular.^2–5 These unique cells were named parietal podocytes and are defined as cells with features of differentiated podocytes lining the inner aspect of Bowman’s capsule (Figure 1A). Using an extended panel of antibodies, Bariety et al. showed that parietal podocytes expressed the same marker proteins as visceral podocytes.¹ On electron microscopic images, parietal podocytes mostly form interdigitating foot processes^3,6,7 and recruit periglomerular capillaries.⁸ In patients with membranous GN, parietal podocytes behaved similar to visceral podocytes in that they formed subepithelial deposits on the parietal basement membrane identical to those on the glomerular basement membrane (GBM).⁵Open in a separate windowFigure 1.Characterization of the early events in the coagulation model. (A) Schematic of a parietal podocyte on Bowman’s capsule forming foot processes (arrow) and recruiting periglomerular capillaries (asterisk). (B and B’) Schematic of the electrocoagulation model. Along the lateral margin of the kidney, a necrotic area is visible after coagulation (arrowheads, right kidney). (C) Immediately after coagulation, a triangular area of necrosis is observed, reaching from the renal cortex into the medulla. (D) Higher magnification of the interface between the coagulated renal tissue (arrow) and remaining parenchyma shows several necrotic tubules 3 days after coagulation (arrowheads) with inflammatory demarcation. (E) After 1 week, multiple tubules are dilated (arrowhead). (F) After 3 weeks, glomeruli with obliterated urinary poles (arrow) and atrophic tubular remnants (arrowheads) are observed. C–F show periodic acid–Schiff stainings of paraffin sections. (G) Immunohistologic double staining shows that cells obstructing the tubular outlet are SSeCKS-positive parietal cells (arrow) and that Bowman’s capsule is lined by parietal podocytes projecting from the vascular pole (arrowheads). (H–I) TUNEL assays consistently show positive nuclear staining in PECs (approximately 1–2 positive nuclei per 100 glomeruli) 1–3 weeks after electrocoagulation (G, arrow). (H) No nuclear staining is observed in healthy controls.The number of parietal podocytes on Bowman’s capsule may vary significantly. Published data indicate that their number increases throughout life.⁹ In normal kidneys, parietal podocytes are rare,¹ whereas their frequency and number is increased in transplant nephrectomies and in diseased kidneys.^2,5 In atubular glomeruli or glomerular cysts, parietal podocytes can often be found on Bowman’s capsule—in many cases, covering its entire circumference.^1,7,10–12 Atubular glomeruli and glomerular cysts are not uncommon even in “normal kidney tissue” obtained from tumor nephrectomies (up to 1% of all glomeruli).^10,13 Both are particularly common, however, in renal diseases affecting the tubulointerstitium.^14–19 Furthermore, atubular/cystic glomeruli have also been observed in other renal diseases,²⁰ including diabetic nephropathy in which on average 17% of all glomeruli were atubular,¹³ transplanted kidney affected by chronic allograft rejection (60% atubular glomeruli),¹⁰ severe renal artery stenosis (50% atubular glomeruli),²¹ GN,^5,10 pyelonephritis,¹⁴ and polycystic kidney disease.^22,23 Glomerular cysts lined by parietal podocytes have also been described in different cystic diseases in animals.^7,11,12,24In previous studies, it was proposed that parietal epithelial cells (PECs) may differentiate into podocytes acting as a potential intrinsic progenitor cell population.^25,26 Parietal podocytes provide a unique and exceptional situation to investigate whether PECs have the potential to transdifferentiate into podocytes in situ on Bowman’s capsule. In this study, we set out to resolve the origin of parietal podocytes by lineage tracing. For this purpose, a novel model to induce the generation of parietal podocytes in atubular glomeruli was established.     

Pregnancy and Maternal Outcomes Among Kidney Transplant Recipients

Fertility rates, pregnancy, and maternal outcomes are not well described among women with a functioning kidney transplant. Using data from the Australian and New Zealand Dialysis and Transplant Registry, we analyzed 40 yr of pregnancy-related outcomes for transplant recipients. This analysis included 444 live births reported from 577 pregnancies; the absolute but not relative fertility rate fell during these four decades. Of pregnancies achieved, 97% were beyond the first year after transplantation. The mean age at the time of pregnancy was 29 ± 5 yr. Compared with previous decades, the mean age during the last decade increased significantly to 32 yr (P < 0.001). The proportion of live births doubled during the last decade, whereas surgical terminations declined (P < 0.001). The fertility rate (or live-birth rate) for this cohort of women was 0.19 (95% confidence interval 0.17 to 0.21) relative to the Australian background population. We also matched 120 parous with 120 nulliparous women by year of transplantation, duration of transplant, age at transplantation ±5 yr, and predelivery creatinine for parous women or serum creatinine for nulliparous women; a first live birth was not associated with a poorer 20-yr graft or patient survival. Maternal complications included preeclampsia in 27% and gestational diabetes in 1%. Taken together, these data confirm that a live birth in women with a functioning graft does not have an adverse impact on graft and patient survival.One of the many perceived benefits of kidney transplantation has been restoration of pituitary-ovarian function and fertility in women of reproductive age. Prenatal advice for women with a functioning kidney transplant has been primarily based on data derived from observational research,^1–13 and the reported live-birth rates achieved in such women range from 43.2¹⁴ to 82%.¹⁵Although an increased pregnancy event number has been reported for women with a functioning kidney transplant,¹⁶ little is actually known about “pregnancy rate changes” during the past 40 yr. More importantly, long-term graft and maternal survival analyses, referred to when advising women who have undergone transplantation and are considering a pregnancy, have been mostly performed without adequate matching,¹² or, alternatively, matching has been used but outcomes followed up for only brief intervals^14,17,18 and in small cohorts.^19–22 Published graft matching studies to date suggest no adverse impact 10 yr after a live birth.¹⁴In most instances, pregnancies in women with a kidney graft have been encouraged. Historically, renal function,^8,15,17,18 baseline proteinuria,²³ intercurrent hypertension,^1,24 and time from transplantation^{1,3,5,8,14,15,18,24,25} have been used to predict adverse event risks to the mother, kidney, and offspring. To this are added the often unquantifiable inherent risks for genetically transmitted diseases or the problems associated with prematurity.^26,27 More recently, epidemiologic evidence suggests low birth weight may be associated with the development of hypertension,²⁸ cardiovascular disease,²⁹ insulin resistance,³⁰ and end-stage renal failure.³¹ Moreover, low birth weight is associated with an increased risk for hypertension, independent of genetic and shared environmental factors.³²Series published to date have not captured all pregnancy events or their outcomes. Limitations of some of the published studies include short duration of follow-up and studies with no adequate or long-term matching for decade and renal function.We examined fertility rates, pregnancy rates, and pregnancy outcomes over 40 yr in an at-risk population, defined as women who were aged between 15 and 49 and had a functioning kidney transplant, using ANZDATA registry data. In addition, maternal and graft outcomes were analyzed, and, uniquely, a matched cohort analysis of 120 nulliparous and 120 parous women who had undergone transplantation enabled analysis of outcomes at 20 yr.     

Albuminuria and Estimated Glomerular Filtration Rate Independently Associate with Acute Kidney Injury

Acute kidney injury (AKI) is increasingly common and a significant contributor to excess death in hospitalized patients. CKD is an established risk factor for AKI; however, the independent graded association of urine albumin excretion with AKI is unknown. We analyzed a prospective cohort of 11,200 participants in the Atherosclerosis Risk in Communities (ARIC) study for the association between baseline urine albumin-to-creatinine ratio and estimated GFR (eGFR) with hospitalizations or death with AKI. The incidence of AKI events was 4.0 per 1000 person-years of follow-up. Using participants with urine albumin-to-creatinine ratios <10 mg/g as a reference, the relative hazards of AKI, adjusted for age, gender, race, cardiovascular risk factors, and categories of eGFR were 1.9 (95% CI, 1.4 to 2.6), 2.2 (95% CI, 1.6 to 3.0), and 4.8 (95% CI, 3.2 to 7.2) for urine albumin-to-creatinine ratio groups of 11 to 29 mg/g, 30 to 299 mg/g, and ≥300 mg/g, respectively. Similarly, the overall adjusted relative hazard of AKI increased with decreasing eGFR. Patterns persisted within subgroups of age, race, and gender. In summary, albuminuria and eGFR have strong, independent associations with incident AKI.It has long been recognized that an episode of acute kidney injury (AKI) can have serious health consequences.^1–4 Even a relatively small degree of renal injury increases a patient''s risk of a prolonged hospital stay, chronic kidney disease (CKD), ESRD, and death.^2,5–10 Over the last 2 decades, the incidence of hospitalized AKI has increased dramatically.^11–14 Precise estimations vary depending on population and method of case identification, but a recent community-based study of AKI estimated the incidence of nondialysis requiring AKI at 522 per 100,000 population per year and dialysis-requiring AKI at 30 per 100,000,¹³ which is well over that of ESRD.¹⁴ This increase in the burden of disease, taken with the associated poor long-term outcomes, has established AKI as a major public health issue.¹⁴Beyond routine supportive care, there exists little established medical therapy for AKI.¹⁵ Many current lines of research are focused on the prevention of AKI. However, few prospective, population-based studies have evaluated the development of AKI.^3,13,16 Hsu et al.,^13,17 along with multiple observational series in various clinical settings, have clearly established older age and CKD as risk factors for AKI.^18–24 Other observed associations with AKI include black race and male gender.^11,18,25 Proteinuria, an established risk factor in the development of cardiovascular disease,^26,27 ESRD,²⁸ and death,²⁹ is less studied in its role in the development of AKI. Hsu and colleagues demonstrated the prospective association of proteinuria with dialysis-requiring AKI; however, the proteinuria classification was binary and based on dipstick measurement.¹⁷ To our knowledge, no study has quantified the independent dose response of albuminuria with AKI hospitalization, including less severe AKI. Our study''s objective was thus to characterize prospectively the association between baseline urine albumin-to-creatinine ratio (UACR) and hospitalizations for AKI, controlling for established and potential risk factors such as CKD, age, and cardiovascular comorbidities.     

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.     

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

RESEARCH DESIGN AND METHODS

We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r^−/− and Gcgr^−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG.

RESULTS

Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR.

CONCLUSIONS

Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.Obesity is an important risk factor for type 2 diabetes, and ∼90% of patients with type 2 diabetes are overweight or obese (1). Among new therapies for type 2 diabetes, peptidyl mimetics of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) stimulate insulin biosynthesis and secretion in a glucose-dependent manner (2,3) and cause modest weight loss in type 2 diabetic patients. The glucose-lowering and antiobesity effects of incretin-based therapies for type 2 diabetes have prompted evaluation of the therapeutic potential of other glucagon-family peptides, in particular oxyntomodulin (OXM). The OXM peptide is generated by post-translational processing of preproglucagon in the gut and is secreted postprandially from l-cells of the jejuno-ileum together with other preproglucagon-derived peptides including GLP-1 (4,5). In rodents, OXM reduces food intake and body weight, increases energy expenditure, and improves glucose metabolism (6–8). A 4-week clinical study in obese subjects demonstrated that repeated subcutaneous administration of OXM was well tolerated and caused significant weight loss with a concomitant reduction in food intake (9). An increase in activity-related energy expenditure was also noted in a separate study involving short-term treatment with the peptide (10).OXM activates both, the GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in vitro, albeit with 10- to 100-fold reduced potency compared with the cognate ligands GLP-1 and glucagon, respectively (11–13). It has been proposed that OXM modulates glucose and energy homeostasis solely by GLP1R agonism, because its acute metabolic effects in rodents are abolished by coadministration of the GLP1R antagonist exendin(9–39) and are not observed in Glp1r^−/− mice (7,8,14,15). Other aspects of OXM pharmacology, however, such as protective effects on murine islets and inhibition of gastric acid secretion appear to be independent of GLP1R signaling (14). In addition, pharmacological activation of GCGR by glucagon, a master regulator of fasting metabolism (16), decreases food intake in rodents and humans (17–19), suggesting a potential role for GCGR signaling in the pharmacology of OXM. Because both OXM and GLP-1 are labile in vivo (T_1/2 ∼12 min and 2–3 min, respectively) (20,21) and are substrates for the cell surface protease dipeptidyl peptidase 4 (DPP-4) (22), we developed two long-acting DPP-4–resistant OXM analogs as pharmacological agents to better investigate the differential pharmacology and therapeutic potential of dual GLP1R/GCGR agonism versus GLP1R-selective agonism. Peptide DualAG exhibits in vitro GLP1R and GCGR agonist potency comparable to that of native OXM and is conjugated to cholesterol via a Cys sidechain at the C-terminus for improved pharmacokinetics. Peptide GLPAG differs from DualAG by only one residue (Gln³→Glu) and is an equipotent GLP1R agonist, but has no significant GCGR agonist or antagonist activity in vitro. The objective of this study was to leverage the matched GLP1R agonist potencies and pharmacokinetics of peptides DualAG and GLPAG in comparing the metabolic effects and therapeutic potential of a dual GLP1R/GCGR agonist with a GLP1R-selective agonist in a mouse model of obesity.     

Anion Exchanger 1 Interacts with Nephrin in Podocytes

The central role of the multifunctional protein nephrin within the macromolecular complex forming the glomerular slit diaphragm is well established, but the mechanisms linking the slit diaphragm to the cytoskeleton and to the signaling pathways involved in maintaining the integrity of the glomerular filter remain incompletely understood. Here, we report that nephrin interacts with the bicarbonate/chloride transporter kidney anion exchanger 1 (kAE1), detected by yeast two-hybrid assay and confirmed by immunoprecipitation and co-localization studies. We confirmed low-level glomerular expression of kAE1 in human and mouse kidneys by immunoblotting and immunofluorescence microscopy. We observed less kAE1 in human glomeruli homozygous for the NPHS1_FinMaj nephrin mutation, whereas kAE1 expression remained unchanged in the collecting duct. We could not detect endogenous kAE1 expression in NPHS1_FinMaj podocytes in primary culture, but heterologous re-introduction of wild-type nephrin into these podocytes rescued kAE1 expression. In kidneys of Ae1^−/− mice, nephrin abundance was normal but its distribution was altered along with the reported kAE1-binding protein integrin-linked kinase (ILK). Ae1^−/− mice had increased albuminuria with glomerular enlargement, mesangial expansion, mesangiosclerosis, and expansion of the glomerular basement membrane. Glomeruli with ILK-deficient podocytes also demonstrated altered AE1 and nephrin expression, further supporting the functional interdependence of these proteins. These data suggest that the podocyte protein kAE1 interacts with nephrin and ILK to maintain the structure and function of the glomerular basement membrane.Anion exchanger 1 (AE1; SLC4A1), an SLC4 bicarbonate transporter family member, is transcribed as an erythroid isoform (eAE1) and a truncated kidney isoform (kAE1) lacking amino acids 1 through 65 in humans.¹ eAE1 comprises the core of the multiprotein complex of integral and peripheral membrane proteins essential to the structural integrity of the red cell membrane, and its bicarbonate/chloride activity is required for gas transport (see reviews^2,3). In the kidney, kAE1 is localized to the basolateral membrane of collecting duct type A intercalated cells. Normal terminal urinary acidification by these cells requires kAE1-mediated bicarbonate reabsorption into the blood. Specific mutations in AE1 usually cause either autosomal dominant hereditary ovalo-spherocytosis or distal renal tubular acidosis (dRTA).⁴ In rare cases of homozygous recessive or compound heterozygous AE1 mutations, both the erythroid and renal phenotypes can manifest in the same individuals.^5–7 In addition to these established roles, low-level AE1 expression has been detected in the glomerulus,^8,9 but its potential function or interactions in the glomerulus are unknown.AE1 possesses a long cytoplasmic N-terminus, a 12- to 14-span transmembrane transporter domain, and a short C-terminal cytoplasmic tail (Supplemental Figure 1). Both the N- and C-terminal domains of kAE1 contain tyrosine residues critical for basolateral targeting,^10,11 which is likely regulated by phosphorylation.¹¹ The N-terminus of kAE1 interacts with integrin-linked kinase (ILK),¹² a protein that binds the cytoplasmic domains of β-integrins and cytoskeleton-associated proteins.^13,14 The kAE1/ILK interaction enhanced kAE1 trafficking to the plasma membrane in HEK293 cells,¹² but deletion of the majority of the ILK-interacting region in kAE1 did not affect its polarized trafficking in MDCK cells.¹¹ Thus, the physiologic importance of the kAE1–ILK interaction in the kidney remains unclear. We searched for proteins that interact with the C-terminus of kAE1, using a yeast two-hybrid screen of a human kidney cDNA library, and identified a novel interaction between kAE1 and nephrin.Nephrin is a single-spanning transmembrane Ig superfamily protein (Supplemental Figure 1) and an integral component of the podocyte slit diaphragm (SD), a structure critical to the glomerular selectivity filter.¹⁵ Mutations or gene targeting of nephrin results in congenital nephrotic syndrome.^16,17 The nephrin extracellular domain contributes to the structural framework of the SD via homo- and heterodimeric interactions with neighboring nephrin polypeptides and nephrin-like homologs Neph1 and Neph2.^15,18–20 The intracellular domain of nephrin contains multiple tyrosine phosphorylation sites and interacts with podocin,^21,22 CD2-associated protein,^23,24 Nck proteins,^25–27 the ion channel TRPC6,^28,29 and adherens junction proteins.^30,31 These interactions anchor the SD complex to the underlying cytoskeleton and participate in signal transduction. Nephrin also forms a multicomponent ternary complex with ILK.³² The proteinuric phenotype of mice with podocyte-specific deletions of ILK and other components of the basally situated ILK/integrin complex^32–35 suggests that SD and basal domain signaling complexes of podocytes cooperate to maintain integrity of the glomerular filtration barrier.In view of the direct associations of ILK with kAE1¹² and nephrin,³² we investigated the physiologic significance of the nephrin/kAE1 interaction. Our studies demonstrate the importance of nephrin for stable kAE1 expression in podocytes and the in vivo interdependence of levels and subcellular localization among kAE1, nephrin, and ILK in podocytes, suggesting a novel role of kAE1 in glomerular function.     

Unraveling the Role of Podocyte Turnover in Glomerular Aging and Injury

Podocyte loss is a major determinant of progressive CKD. Although recent studies showed that a subset of parietal epithelial cells can serve as podocyte progenitors, the role of podocyte turnover and regeneration in repair, aging, and nephron loss remains unclear. Here, we combined genetic fate mapping with highly efficient podocyte isolation protocols to precisely quantify podocyte turnover and regeneration. We demonstrate that parietal epithelial cells can give rise to fully differentiated visceral epithelial cells indistinguishable from resident podocytes and that limited podocyte renewal occurs in a diphtheria toxin model of acute podocyte ablation. In contrast, the compensatory programs initiated in response to nephron loss evoke glomerular hypertrophy, but not de novo podocyte generation. In addition, no turnover of podocytes could be detected in aging mice under physiologic conditions. In the absence of podocyte replacement, characteristic features of aging mouse kidneys included progressive accumulation of oxidized proteins, deposits of protein aggregates, loss of podocytes, and glomerulosclerosis. In summary, quantitative investigation of podocyte regeneration in vivo provides novel insights into the mechanism and capacity of podocyte turnover and regeneration in mice. Our data reveal that podocyte generation is mainly confined to glomerular development and may occur after acute glomerular injury, but it fails to regenerate podocytes in aging kidneys or in response to nephron loss.Chronic loss of kidney function is of significant public health importance not only because of high prevalence but also because it is a major independent risk factor for cardiovascular morbidity and mortality.¹ Aged kidneys display an increased susceptibility for progressive diseases, suggesting that overlapping molecular programs contribute to organ aging and disease progression.² Recent data indicate that a decrease in the number of glomerular podocytes is an important predictor for kidney aging, and podocytes are considered the weak link in the progression of CKD.³ Animal models have shown that podocyte depletion of up to 20% can be tolerated before a scarring response takes place,⁴ but residual podocytes are unable to undergo cell division.^5–7 Although it is widely believed that glomeruli have only a limited capacity to resolve lesions, cases of potential podocyte regeneration and disease reversal have been described.^8–10 These recent observations have instigated hope of finding ways to stimulate kidney regeneration. So far, two potential podocyte progenitor niches, bone marrow cells and parietal epithelial cells, have been characterized.^11–14 Bone marrow transplantation has an ameliorating effect in several animal models of glomerular disease,^15–18 but the underlying mechanisms remain poorly understood. In humans, Y-chromosome–positive podocytes have been detected in kidneys transplanted from females to males.¹² Located directly adjacent to podocytes, parietal epithelial cells express the stem cell markers CD24 and CD133 in human tissue and are capable of self-renewal, as well as differentiation into several cell types, including podocytes.^11,14,19In this study, a novel flow cytometry–based quantitative method was devised to assess the regenerative capacity of podocytes during diphtheria toxin (DT)–mediated acute podocyte loss, models of chronic nephron loss, and kidney aging.     

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.