Zinc finger protein 24-dependent transcription factor SOX9 up-regulation protects tubular epithelial cells during acute kidney injury.

Transcriptional profiling studies have identified several protective genes upregulated in tubular epithelial cells during acute kidney injury (AKI). Identifying upstream transcriptional regulators could lead to the development of therapeutic strategies augmenting the repair processes. SOX9 is a transcription factor controlling cell-fate during embryonic development and adult tissue homeostasis in multiple organs including the kidneys. SOX9 expression is low in adult kidneys; however, stress conditions can trigger its transcriptional upregulation in tubular epithelial cells. SOX9 plays a protective role during the early phase of AKI and facilitates repair during the recovery phase. To identify the upstream transcriptional regulators that drive SOX9 upregulation in tubular epithelial cells, we used an unbiased transcription factor screening approach. Preliminary screening and validation studies show that zinc finger protein 24 (ZFP24) governs SOX9 upregulation in tubular epithelial cells. ZFP24, a Cys2-His2 (C2H2) zinc finger protein, is essential for oligodendrocyte maturation and myelination; however, its role in the kidneys or in SOX9 regulation remains unknown. Here, we found that tubular epithelial ZFP24 gene ablation exacerbated ischemia, rhabdomyolysis, and cisplatin-associated AKI. Importantly, ZFP24 gene deletion resulted in suppression of SOX9 upregulation in injured tubular epithelial cells. Chromatin immunoprecipitation and promoter luciferase assays confirmed that ZFP24 bound to a specific site in both murine and human SOX9 promoters. Importantly, CRISPR/Cas9-mediated mutation in the ZFP24 binding site in the SOX9 promoter in vivo led to suppression of SOX9 upregulation during AKI. Thus, our findings identify ZFP24 as a critical stress-responsive transcription factor protecting tubular epithelial cells through SOX9 upregulation.

Essential role of Wtip in mouse development and maintenance of the glomerular filtration barrier.

Wilms' tumor interacting protein (Wtip) has been implicated in cell junction assembly and cell differentiation and interacts with proteins in the podocyte slit diaphragm, where it regulates podocyte phenotype. To define Wtip expression and function in the kidney, we created a Wtip-deleted mouse model using ß-galactosidase-neomycin (ß-geo) gene trap technology. Wtip gene trap mice were embryonic lethal, suggesting additional developmental roles outside kidney function. Using ß-geo heterozygous and normal mice, Wtip expression was identified in the developing kidneys, heart, and eyes. In the kidney, expression was restricted to podocytes, which appeared initially at the capillary loop stage coinciding with terminal podocyte differentiation. Heterozygous mice had an expected lifespan and showed no evidence of proteinuria or glomerular pathology. However, heterozygous mice were more susceptible to glomerular injury than wild-type littermates and developed more significant and prolonged proteinuria in response to lipopolysaccharide or adriamycin. In normal human kidneys, WTIP expression patterns were consistent with observations in mice and were lost in glomeruli concurrent with loss of synaptopodin expression in disease. Mechanistically, we identified the Rho guanine nucleotide exchange factor 12 (ARHGEF12) as a binding partner for WTIP. ARHGEF12 was expressed in human podocytes and formed high-affinity interactions through their LIM- and PDZ-binding domains. Our findings suggest that Wtip is essential for early murine embryonic development and maintaining normal glomerular filtration barrier function, potentially regulating slit diaphragm and foot process function through Rho effector proteins.NEW & NOTEWORTHY This study characterized dynamic expression patterns of Wilms' tumor interacting protein (Wtip) and demonstrates the novel role of Wtip in murine development and maintenance of the glomerular filtration barrier.

Molecular profiling of kidney compartments from serial biopsies differentiate treatment responders from non-responders in lupus nephritis.

The immune pathways that define treatment response and non-response in lupus nephritis (LN) are unknown. To characterize these intra-kidney pathways, transcriptomic analysis was done on protocol kidney biopsies obtained at flare (initial biopsy (Bx1)) and after treatment (second biopsy (Bx2)) in 58 patients with LN. Glomeruli and tubulointerstitial compartments were isolated using laser microdissection. RNA was extracted and analyzed by nanostring technology with transcript expression from clinically complete responders, partial responders and non-responders compared at Bx1 and Bx2 and to the healthy controls. Top transcripts that differentiate clinically complete responders from non-responders were validated at the protein level by confocal microscopy and urine ELISA. At Bx1, cluster analysis determined that glomerular integrin, neutrophil, chemokines/cytokines and tubulointerstitial chemokines, T cell and leukocyte adhesion genes were able to differentiate non-responders from clinically complete responders. At Bx2, glomerular monocyte, extracellular matrix, and interferon, and tubulointerstitial interferon, complement, and T cell transcripts differentiated non-responders from clinically complete responders. Protein analysis identified several protein products of overexpressed glomerular and tubulointerstitial transcripts at LN flare, recapitulating top transcript findings. Urine complement component 5a and fibronectin-1 protein levels reflected complement and fibronectin expression at flare and after treatment. Thus, transcript analysis of serial LN kidney biopsies demonstrated how gene expression in the kidney changes with clinically successful and unsuccessful therapy. Hence, these insights into the molecular landscape of response and non-response may help align LN management with the pathogenesis of kidney injury.

ApoL1 Overexpression Drives Variant-Independent Cytotoxicity.

Coding variants in the APOL1 gene are associated with kidney diseases in African ancestral populations; yet, the underlying biologic mechanisms remain uncertain. Variant-dependent autophagic and cytotoxic cell death have been proposed as pathogenic pathways mediating kidney injury. To examine this possibility, we conditionally expressed APOL1-G0 (reference), -G1, and -G2 (variants) using a tetracycline-regulated system in HEK293 cells. Autophagy was monitored biochemically and cell death was measured using multiple assays. We measured intracellular Na+ and K+ content with atomic absorption spectroscopy and APOL1-dependent currents with whole-cell patch clamping. Neither reference nor variant APOL1s induced autophagy. At high expression levels, APOL1-G0, -G1, and -G2 inserted into the plasma membrane and formed pH-sensitive cation channels, causing collapse of cellular Na+ and K+ gradients, phosphorylation of p38 mitogen-activated protein kinase, and cell death, without variant-dependent differences. APOL1-G0 and -G2 exhibited similar channel properties in whole-cell patch clamp experiments. At low expression levels, neither reference nor variant APOL1s localized on the plasma membrane, Na+ and K+ gradients were maintained, and cells remained viable. Our results indicate that APOL1-mediated pore formation is critical for the trypanolytic activity of APOL1 and drives APOL1-mediated cytotoxicity in overexpression systems. The absence of cytotoxicity at physiologic expression levels suggests variant-dependent intracellular K+ loss and cytotoxicity does not drive kidney disease progression.

Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2).

Albuminuria and tubular atrophy are among the highest risks for CKD progression to ESRD. A parsimonious mechanism involves leakage of albumin-bound nonesterified fatty acids (NEFAs) across the damaged glomerular filtration barrier and subsequent reabsorption by the downstream proximal tubule, causing lipoapoptosis. We sought to identify the apical proximal tubule transporter that mediates NEFA uptake and cytotoxicity. We observed transporter-mediated uptake of fluorescently labeled NEFA in cultured proximal tubule cells and microperfused rat proximal tubules, with greater uptake from the apical surface than from the basolateral surface. Protein and mRNA expression analyses revealed that kidney proximal tubules express transmembrane fatty acid transporter-2 (FATP2), encoded by Slc27a2, but not the other candidate transporters CD36 and free fatty acid receptor 1. Kidney FATP2 localized exclusively to proximal tubule epithelial cells along the apical but not the basolateral membrane. Treatment of mice with lipidated albumin to induce proteinuria caused a decrease in the proportion of tubular epithelial cells and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a2-/- mice. Ex vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic apical proximal tubule exposure during glomerular injury revealed significantly reduced NEFA uptake and palmitate-induced apoptosis in microperfused Slc27a2-/- proximal tubules and Slc27a2-/- or FATP2 shRNA-treated proximal tubule cell lines compared with wild-type or scrambled oligonucleotide-treated cells, respectively. We conclude that FATP2 is a major apical proximal tubule NEFA transporter that regulates lipoapoptosis and may be an amenable target for the prevention of CKD progression.

APOL1-G0 or APOL1-G2 Transgenic Models Develop Preeclampsia but Not Kidney Disease.

APOL1 risk variants are associated with kidney disease in blacks, but the mechanisms of renal injury associated with APOL1 risk variants are unknown. Because APOL1 is unique to humans and some primates, we created transgenic (Tg) mice using the promoter of nephrin-encoding Nphs1 to express the APOL1 reference sequence (G0) or the G2 risk variant in podocytes, establishing Tg lines with a spectrum of APOL1 expression levels. Podocytes from Tg-G0 and Tg-G2 mice did not undergo necrosis, apoptosis, or autophagic cell death in vivo, even in lines with highly expressed transgenes. Further, Tg-G0 and Tg-G2 mice did not develop kidney pathology, proteinuria, or azotemia as of 300 days of age. However, by 200 days of age, Tg-G2 mice had significantly lower podocyte density than age-matched WT and Tg-G0 mice had, a difference that was not evident at weaning. Notably, a pregnancy-associated phenotype that encompassed eclampsia, preeclampsia, fetal/neonatal deaths, and small litter sizes occurred in some Tg-G0 mice and more severely in Tg-G2 mice. Similar to human placenta, placentas of Tg mice expressed APOL1. Overall, these results suggest podocyte depletion could predispose individuals with APOL1 risk genotypes to kidney disease in response to a second stressor, and add to other published evidence associating APOL1 expression with preeclampsia.

Perioperative Follow-Up of Patients With Severe Pulmonary Artery Hypertension Secondary to Left Heart Disease: A Single Center, Prospective, Observational Study.

OBJECTIVE: A substantial portion of the Indian cardiac surgery population experiences rheumatic valve disease that progresses to severe pulmonary artery hypertension (PAH) in a few patients. Right ventricular (RV) function, particularly in the perioperative period, has been studied sparsely. The authors describe serial RV function and clinical variables in the perioperative period in patients with severe PAH secondary to left heart disease. DESIGN: Prospective, observational study. SETTING: University hospital. PARTICIPANTS: Patients with PAH. INTERVENTIONS: The study comprised consecutive patients referred for open cardiac surgery from January 2012 to June 2013 who also had an estimated right ventricular systolic pressure≥50 mmHg on referral echocardiogram. Composite echocardiographic assessment of right ventricular size and linear/two-dimensional tissue Doppler systolic function and diastolic function analysis were performed at predetermined intervals. Data from right heart catheterization, inotrope use, fluid requirements, mechanical ventilation logs, and intensive care stay also were acquired. MEASUREMENTS AND MAIN RESULTS: A complete dataset was obtained in 20 of 22 patients enrolled in the study. Serial comparison of most RV echocardiographic function variables were noted to be abnormal at baseline, deteriorating further in the immediate postoperative period and trending to a partial recovery at discharge from the intensive care unit, particularly for longitudinal assessment of the RV. Fractional area change, although abnormal, was noted to be preserved. Pulmonary artery systolic pressures registered significantly declined after intervention. The clinical course was largely uneventful. CONCLUSION: Although linear echocardiographic RV function was grossly abnormal in the perioperative period in this patient subset with PAH, there was apparent disjunction with the clinical course.

Plasma apolipoprotein L1 levels do not correlate with CKD.

Polymorphisms in APOL1 are associated with CKD, including HIV-related CKD, in individuals of African ancestry. The apolipoprotein L1 (APOL1) protein circulates and is localized in kidney cells, but the contribution of APOL1 location to CKD pathogenesis is unclear. We examined associations of plasma APOL1 levels with plasma cytokine levels, dyslipidemia, and APOL1 genotype in a nested case-control study (n=270) of HIV-infected African Americans enrolled in a multicenter prospective observational study. Patients were designated as having CKD when estimated GFR (eGFR) decreased to <60 ml/min per 1.73 m(2) (eGFR<60 cohort) or protein-to-creatinine ratios became >3.5 g/g (nephrotic proteinuria cohort). Circulating APOL1 levels did not associate with APOL1 genotype, CKD status, or levels of proinflammatory cytokines, but did correlate with fasting cholesterol, LDL cholesterol, and triglyceride levels. At ascertainment, CKD-associated polymorphisms (risk variants) in APOL1 associated with the eGFR<60 cohort, but not the nephrotic-range proteinuria cohort. Of note, in both the eGFR<60 and nephrotic proteinuria cohorts, CKD cases with two APOL1 risk variants had significant declines in eGFR over a median of 4 years compared with individuals with one or no risk variants. APOL1 risk genotype was not associated with changes in proteinuria. Higher circulating proinflammatory cytokine levels were independently associated with CKD but not APOL1 genotype. In conclusion, the function of variant APOL1 proteins derived from circulation or synthesized in the kidney, but not the level of circulating APOL1, probably mediates APOL1-associated kidney disease in HIV-infected African Americans.

The biology of APOL1 with insights into the association between APOL1 variants and chronic kidney disease.

Recent studies have identified genetic variants in APOL1 that may contribute to the increased incidence of kidney disease in populations with African ancestry. Here, we review the biology of APOL1 present in the circulation and localized to the kidney as it may contribute to the pathogenesis of APOL1-associated kidney disease.

Characterization of Glomerular and Tubulointerstitial Proteomes in a Case of Nonsteroidal Anti-Inflammatory Drug-Attributed Acute Kidney Injury: A Clinical Pathologic Molecular Correlation.

The major goals of the Kidney Precision Medicine Project (KPMPP) are to establish a molecular atlas of the kidney in health and disease and improve our understanding of the molecular drivers of CKD and AKI. In this clinical-pathologic-molecular correlation, we describe the case of a 38-year-old woman without any history of CKD who underwent a research kidney biopsy in the setting of AKI suspected to be due to nonsteroidal anti-inflammatory use after cesarean section delivery. The participant's histopathology was consistent with mild acute tubular injury, without significant interstitial fibrosis or tubular atrophy. This diagnosis was supported by analysis of the glomerular and tubulointerstitial proteomes. The proteomic interrogation revealed a molecular landscape that demonstrated differences in kidney prostaglandin synthesis that may be in response to nonsteroidal anti-inflammatory drugs and signs of intrarenal inflammation and fibrosis that were not evident by histopathology alone.

Deletion of NuRD component Mta2 in nephron progenitor cells causes developmentally programmed FSGS.

Low nephron endowment at birth is a risk factor for chronic kidney disease. The prevalence of this condition is increasing due to higher survival rates of preterm infants and children with multi- organ birth defect syndromes that affect the kidney and urinary tract. We created a mouse model of congenital low nephron number due to deletion of Mta2 in nephron progenitor cells. Mta2 is a core component of the Nucleosome Remodeling and Deacetylase (NuRD) chromatin remodeling complex. These mice developed albuminuria at 4 weeks of age followed by focal segmental glomerulosclerosis (FSGS) at 8 weeks, with progressive kidney injury and fibrosis. Our studies reveal that altered mitochondrial metabolism in the post-natal period leads to accumulation of neutral lipids in glomeruli at 4 weeks of age followed by reduced mitochondrial oxygen consumption. We found that NuRD cooperated with Zbtb7a/7b to regulate a large number of metabolic genes required for fatty acid oxidation and oxidative phosphorylation. Analysis of human kidney tissue also supported a role for reduced mitochondrial lipid metabolism and ZBTB7A/7B in FSGS and CKD. We propose that an inability to meet the physiological and metabolic demands of post-natal somatic growth of the kidney promotes the transition to CKD in the setting of glomerular hypertrophy due to low nephron endowment.

WT1-interacting protein (Wtip) regulates podocyte phenotype by cell-cell and cell-matrix contact reorganization.

Podocytes respond to environmental cues by remodeling their slit diaphragms and cell-matrix adhesive junctions. Wt1-interacting protein (Wtip), an Ajuba family LIM domain scaffold protein expressed in the podocyte, coordinates cell adhesion changes and transcriptional responses to regulate podocyte phenotypic plasticity. We evaluated effects of Wtip on podocyte cell-cell and cell-matrix contact organization using gain-of- and loss-of-function methods. Endogenous Wtip targeted to focal adhesions in adherent but isolated podocytes and then shifted to adherens junctions after cells made stable, homotypic contacts. Podocytes with Wtip knockdown (shWtip) adhered but failed to spread normally. Noncontacted shWtip podocytes did not assemble actin stress fibers, and their focal adhesions failed to mature. As shWtip podocytes established cell-cell contacts, stable adherens junctions failed to form and F-actin structures were disordered. In shWtip cells, cadherin and ß-catenin clustered in irregularly distributed spots that failed to laterally expand. Cell surface biotinylation showed diminished plasma membrane cadherin, ß-catenin, and α-catenin in shWtip podocytes, although protein expression was similar in shWtip and control cells. Since normal actin dynamics are required for organization of adherens junctions and focal adhesions, we determined whether Wtip regulates F-actin assembly. Undifferentiated podocytes did not elaborate F-actin stress fibers, but when induced to overexpress WTIP, formed abundant stress fibers, a process blocked by the RhoA inhibitor C3 toxin and a RhoA kinase inhibitor. WTIP directly interacted with Rho guanine nucleotide exchange factor (GEF) 12 (Arhgef12), a RhoA-specific GEF enriched in the glomerulus. In conclusion, stable assembly of podocyte adherens junctions and cell-matrix contacts requires Wtip, a process that may be mediated by spatiotemporal regulation of RhoA activity through appropriate targeting of Arhgef12.

APOL1 localization in normal kidney and nondiabetic kidney disease.

In patients of African ancestry, genetic variants in APOL1, which encodes apolipoprotein L1, associate with the nondiabetic kidney diseases, focal segmental glomerulosclerosis (FSGS), HIV-associated nephropathy (HIVAN), and hypertensive nephropathy. Understanding the renal localization of APOL1 may provide clues that will ultimately help elucidate the mechanisms by which APOL1 variants promote nephropathy. Here, we used immunohistology to examine APOL1 localization in normal human kidney sections and in biopsies demonstrating either FSGS (n = 8) or HIVAN (n = 2). Within normal glomeruli, APOL1 only localized to podocytes. Compared with normal glomeruli, fewer cells stained for APOL1 in FSGS and HIVAN glomeruli, even when expression of the podocyte markers GLEPP1 and synaptopodin appeared normal. APOL1 localized to proximal tubular epithelia in normal kidneys, FSGS, and HIVAN. We detected APOL1 in the arteriolar endothelium of normal and diseased kidney sections. Unexpectedly, in both FSGS and HIVAN but not normal kidneys, the media of medium artery and arterioles contained a subset of α-smooth muscle actin-positive cells that stained for APOL1. Comparing the renal distribution of APOL1 in nondiabetic kidney disease to normal kidney suggests that a previously unrecognized arteriopathy may contribute to disease pathogenesis in patients of African ancestry.

Out on a LIM: chronic kidney disease, podocyte phenotype and the Wilm's tumor interacting protein (WTIP).

Normal function of the glomerular filtration barrier requires wild-type differentiation of the highly specialized glomerular epithelial cell, the podocyte. Podocytes express three distinct domains, consisting of a cell body, primary processes, and secondary foot processes (FP). These FP express slit diaphragms, which are highly specialized cell-cell contacts critical for filtration-barrier function. Foot processes are dynamic structures that reorganize within minutes through actin cytoskeletal rearrangement. Glomerular diseases are characterized by a persistent simplification in podocyte domain structure with loss of FP, a phenotype described as FP effacement. The generation of such phenotypic plasticity requires that signaling pathways in subcellular compartments be integrated dynamically for a cell to respond appropriately to information flow from its microenvironment. We have identified a LIM-domain-containing protein, Wilm's tumor interacting protein (WTIP), that regulates podocyte actin dynamics to maintain stable cell contacts. After glomerular injury, the WTIP molecule shuttles to the podocyte nucleus in response to changes in slit-diaphragm assembly, and changes gene transcription to permit podocyte remodeling. Defining regulatory pathways of podocyte differentiation identifies novel, druggable targets for chronic kidney diseases characterized by glomerular scarring.

The Relationship between APOL1 Structure and Function: Clinical Implications.

Common variants in the APOL1 gene are associated with an increased risk of nondiabetic kidney disease in individuals of African ancestry. Mechanisms by which APOL1 variants mediate kidney disease pathogenesis are not well understood. Amino acid changes resulting from the kidney disease-associated APOL1 variants alter the three-dimensional structure and conformational dynamics of the C-terminal α-helical domain of the protein, which can rationalize the functional consequences. Understanding the three-dimensional structure of the protein, with and without the risk variants, can provide insights into the pathogenesis of kidney diseases mediated by APOL1 variants.

Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys.

The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases.

Establishing a Case for Anti-complement Therapy in Membranous Nephropathy.

INTRODUCTION: Membranous nephropathy (MN) is a common cause of adult nephrotic syndrome that progresses to end-stage kidney disease in up to 40% of cases. It is an autoimmune disease characterized by glomerular subepithelial deposits containing IgG. In experimental MN, these deposits activate complement and cause kidney damage. The role of complement in human MN is less clearly defined. To address this, the current study focused on the role of complement in 2 independent primary (p) MN cohorts. METHODS: Glomeruli were isolated by laser capture microdissection and analyzed by mass spectrometry, focusing on complement proteins, from kidney biopsy specimens from a pMN cohort (n = 11) and from normal controls (n = 5). Immunohistological staining of kidney biopsy specimens for complement proteins was also done. In a second pMN cohort (n = 13), urine levels of Ba, C5a, and C5b-9 (membrane attack complex [MAC]) were measured. RESULTS: Mass spectrometry identified 8 complement pathway components (C1q, C3, C4, C5, C6, C7, C8, and C9) and 5 complement regulators (complement receptor type 1 [CR1], factor H [FH], FH-related protein 2 [FHR2], vitronectin, and clusterin). All complement levels were significantly higher in the MN groups than in the control group, except the level of CR1, which was lower. All pMN biopsy specimens showed negative or trace staining for C1q, positive staining for C3 and C4, and positive staining for at least 1 component of the lectin pathway. Urine Ba, C5a, and MAC were present in pMN, and their levels correlated (r Ba,C5a = 0.87, r Ba,MAC = 0.89, and r C5a,MAC = 0.97, P = .001 for each correlation). CONCLUSION: Elevated glomerular levels of C3, C4, and components of MAC (C5b-9) and absent or decreased levels of the complement regulator CR1, along with increased levels of complement activation products in the urine, support the involvement of complement in the pathogenesis of MN. These data raise the possibility that anti-complement therapies may be effective in some forms of MN.