Glomerular Elasticity and Gene Expression Patterns Define Two Phases of Alport Nephropathy.

Rationale: We take a unique approach to understanding the causes of podocyte injury in collagen IV nephropathies, a crucial step in developing targeted therapies for conditions like Alport Syndrome. Objectives: We characterize the structural, functional, and biophysical properties of glomerular capillaries and podocytes in Col4α3 -/- mice and analyze kidney cortex transcriptional profiles at various disease stages. We investigate the effects of the ER stress mitigator TUDCA on these parameters. Furthermore, we used human FSGS associated podocyte enriched genes to identify molecular pathways rescued by TUDCA thereby offering potential therapeutic targets for Alport Syndrome. Findings: We find a clear disease progression timeline in Col4α3 -/- mice. Podocyte injury develops by 3 months, with glomeruli reaching maximum deformability at 4 months, associated with a 40% loss of podocytes. This is followed by progressive stiffening of glomerular capillaries, increasing proteinuria, reduced renal function, inflammatory infiltrates, and fibrosis from months 4 to 8. Bulk RNA sequencing at 2, 4, and 7 months reveals a progressive increase in expression of genes related to cytokine and chemokine signaling, matrix and cell injury, and activation of the TNF pathway, similar to observations in a NEPTUNE FSGS cohort. Podocyte-enriched genes from FSGS patients mapped to mice found that TUDCA, which mitigated glomerular and renal injury suppressed molecular pathways associated with extracellular matrix and basement membrane synthesis, podocyte stress and hypertrophy. Conclusions: We uncover two distinct phases of Col4α3 -/- nephropathy progression. The first is characterized by podocytopathy, increased glomerular capillary deformability and accelerated podocyte loss, and the second by increased capillary wall stiffening and renal inflammatory and profibrotic pathway activation. The response of podocytes to TUDCA treatment provides novel insights into downstream signaling pathways, offering potential therapeutic targets for treating Alport and related nephropathies.

Fetal High-Risk APOL1 Genotype Increases Risk for Small for Gestational Age in Term Infants Affected by Preeclampsia.

BACKGROUND: Hypertensive disorders of pregnancy cause fetal growth restriction and increased maternal morbidity and mortality, especially in women of African ancestry. Recently, preeclampsia risk was associated with polymorphisms in the apolipoprotein L1 (APOL1) gene in women of African ancestry. OBJECTIVES: We assessed APOL1 genotype effects on pregnancies with and without preeclampsia. METHOD: We conducted an unmatched case-control study of 1,358 mother-infant pairs from two independent cohorts of black women. RESULTS: Term preeclampsia cases with high-risk APOL1 genotypes were more likely to be small for gestational age compared to APOL1 low-risk term cases (odds ratio [OR] 2.8) and APOL1 high-risk controls (OR 5.5). Among preterm pregnancies, fetal APOL1 genotype was associated with preeclampsia. CONCLUSIONS: Fetal APOL1 genotype was associated with preeclampsia in preterm infants and with altered fetal growth in term infants. This may indicate APOL1 genotype impacts a spectrum of pregnancy complications mediated by a common pathophysiological event of placental insufficiency.

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.

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.

Apolipoprotein L1 and mechanisms of kidney disease susceptibility.

PURPOSE OF REVIEW: Allelic variants in the gene for apolipoprotein L1 (APOL1), found only in individuals of African ancestry, explain a majority of the excess risk of kidney disease in African Americans. However, a clear understanding how the disease-associated APOL1 variants cause kidney injury and the identity of environmental stressors that trigger the injury process have not been determined. RECENT FINDINGS: Basic mechanistic studies of APOL1 biochemistry and cell biology, bolstered by new antibody reagents and inducible pluripotent stem cell-derived cell systems, have focused on the cytotoxic effect of the risk variants when APOL1 gene expression is induced. Since the APOL1 variants evolved to alter a key protein-protein interaction with the trypanosome serum resistance-associated protein, additional studies have begun to address differences in APOL1 interactions with other proteins expressed in podocytes, including new observations that APOL1 variants may alter podocyte cytoskeleton dynamics. SUMMARY: A unified mechanism of pathogenesis for the various APOL1 nephropathies still remains unclear and controversial. As ongoing studies have consistently implicated the pathogenic gain-of-function effects of the variant proteins, novel therapeutic development inhibiting the synthesis or function of APOL1 proteins is moving toward clinical trials.

Podocyte density is reduced in kidney allografts with high-risk APOL1 genotypes at transplantation.

Variants in apolipoprotein L1 (APOL1) gene are associated with nondiabetic kidney diseases in black subjects and reduced kidney transplant graft survival. Living and deceased black kidney donors (n = 107) were genotyped for APOL1 variants. To determine whether allografts from high-risk APOL1 donors have reduced podocyte densities contributing to allograft failure, we morphometrically estimated podocyte number, glomerular volume, and podocyte density. We compared allograft loss and eGFR trajectories stratified by APOL1 high-risk and low-risk genotypes. Demographic characteristics were similar in high-risk (n = 16) and low-risk (n = 91) donors. Podocyte density was significantly lower in high-risk than low-risk donors (108 ± 26 vs 127 ± 40 podocytes/106 um3 , P = .03). Kaplan-Meier graft survival (high-risk 61% vs. low-risk 91%, p-value = 0.049) and multivariable Cox models (hazard ratio = 2.6; 95% CI, 0.9-7.8) revealed higher graft loss in recipients of APOL1 high-risk allografts over 48 months. More rapid eGFR decline was seen in recipients of high-risk APOL1 allografts (P < .001). At 60 months, eGFR was 27 vs. 51 mL/min/1.73 min2 in recipients of APOL1 high-risk vs low-risk kidney allografts, respectively. Kidneys from high-risk APOL1 donors had worse outcomes versus low-risk APOL1 genotypes. Lower podocyte density in kidneys from high-risk APOL1 donors may increase susceptibility to CKD from subsequent stresses in both the recipients and donors.

Association of preeclampsia with infant APOL1 genotype in African Americans.

BACKGROUND: Black women in the United States and Africa are at an increased risk for preeclampsia. Allelic variants in the gene for apolipoprotein LI, APOL1, are found only in populations of African ancestry, and have been shown to contribute significant risk for kidney disease. Recent studies suggest these APOL1 variants also may contribute risk for preeclampsia. METHODS: The association of preeclampsia with carriage of APOL1 risk alleles was evaluated in a case-control study of deliveries from black women at a single center in Cleveland, Ohio that included gross and histopathologic evaluations of placental tissues (395 cases and 282 controls). Using logistic regression models, associations between fetal APOL1 genotype and preeclampsia were evaluated using several case definitions based on prematurity and severity of preeclampsia, with uncomplicated term pregnancies as controls. Associations between APOL1 genotype and pathological features were also examined. RESULTS: The infant APOL1 genotype was significantly associated with preeclampsia in a dominant inheritance pattern with odds ratio of 1.41 (P=0.029, 95% CI 1.037, 1.926). Stratifying preeclampsia cases by preterm birth, significant associations were detected for both recessive (O.R.=1.70, P=0.038) and additive (O.R.=1.33, P=0.028) inheritance patterns. APOL1 genotype, however, was not significantly associated with pathological changes or other perinatal observations. CONCLUSIONS: Preeclampsia appears to be another disease associated with APOL1 variants, however, further studies are needed to increase confidence in the mode of inheritance. By understanding the association of APOL1 variants with preeclampsia, genetic screening tests for APOL1 may be useful to predict at-risk pregnancies and targeted interventions may be developed to improve pregnancy outcomes.

APOL1-G0 protects podocytes in a mouse model of HIV-associated nephropathy.

African polymorphisms in the gene for Apolipoprotein L1 (APOL1) confer a survival advantage against lethal trypanosomiasis but also an increased risk for several chronic kidney diseases (CKD) including HIV-associated nephropathy (HIVAN). APOL1 is expressed in renal cells, however, the pathogenic events that lead to renal cell damage and kidney disease are not fully understood. The podocyte function of APOL1-G0 versus APOL1-G2 in the setting of a known disease stressor was assessed using transgenic mouse models. Transgene expression, survival, renal pathology and function, and podocyte density were assessed in an intercross of a mouse model of HIVAN (Tg26) with two mouse models that express either APOL1-G0 or APOL1-G2 in podocytes. Mice that expressed HIV genes developed heavy proteinuria and glomerulosclerosis, and had significant losses in podocyte numbers and reductions in podocyte densities. Mice that co-expressed APOL1-G0 and HIV had preserved podocyte numbers and densities, with fewer morphologic manifestations typical of HIVAN pathology. Podocyte losses and pathology in mice co-expressing APOL1-G2 and HIV were not significantly different from mice expressing only HIV. Podocyte hypertrophy, a known compensatory event to stress, was increased in the mice co-expressing HIV and APOL1-G0, but absent in the mice co-expressing HIV and APOL1-G2. Mortality and renal function tests were not significantly different between groups. APOL1-G0 expressed in podocytes may have a protective function against podocyte loss or injury when exposed to an environmental stressor. This was absent with APOL1-G2 expression, suggesting APOL1-G2 may have lost this protective function.

Common Mechanisms of Viral Injury to the Kidney.

Viral infections in an immunocompetent host can cause both acute and chronic kidney diseases, either by direct damage to the infected kidney cells or as a consequence of systemic immune responses that impact the kidneys' function. Viruses have evolved mechanisms to hijack signaling pathways of the infected cell, including the mammalian target of rapamycin pathway to support viral replication, and to evade antiviral immune responses such as those mediated by miR-155 via microRNA mimetics expressed by the virus. At both the cellular and systemic levels, the host has also evolved mechanisms to counter the viral subversion strategies in the evolutionary battle for mutual survival. In the era of genomic medicine, understanding individual genetic variations that lead to differences in susceptibilities to infection and variabilities in immune responses may open new avenues for treatment, such as the recently described functions of apolipoprotein L1 risk alleles in HIV-associated nephropathy. In addition, state-of-the-art high-throughput sequencing methods have discovered new viruses as the cause for chronic diseases not previously attributed to an infection. The potential application of these methods to idiopathic kidney diseases may reveal similar occult infections by unknown viruses. Precision medicine objectives to optimize host-directed and pathogen-directed therapies for kidney diseases associated with infectious causes will only be achieved through detailed understanding of genetic susceptibility associated with immune responses and viral tropism.

APOL1 polymorphisms and kidney disease: loss-of-function or gain-of-function?

The mechanism that explains the association of APOL1 variants with nondiabetic kidney diseases in African Americans remains unclear. Kidney disease risk is inherited as a recessive trait, and many studies investigating the intracellular function of APOL1 have indicated the APOL1 variants G1 and G2 are associated with cytotoxicity. Whether cytotoxicity results from the absence of a protective effect conferred by the G0 allele or is induced by a deleterious effect of variant allele expression has not be conclusively established. A central issue hampering basic biology studies is the lack of model systems that authentically replicate APOL1 expression patterns. APOL1 is present in humans and a few other primates and appears to have important functions in the kidney, as the kidney is the primary target for disease associated with the genetic variance. There have been no studies to date assessing the function of untagged APOL1 protein under native expression in human or primate kidney cells, and no studies have examined the heterozygous state, a disease-free condition in humans. A second major issue is the chronic kidney disease (CKD)-associated APOL1 variants are conditional mutations, where the disease-inducing function is only evident under the appropriate environmental stimulus. In addition, it is possible there may be more than one mechanism of pathogenesis that is dependent on the nature of the stressor or other genetic variabilities. Studies addressing the function of APOL1 and how the CKD-associated APOL1 variants cause kidney disease are challenging and remain to be fully investigated under conditions that faithfully model known human genetics and physiology.

Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models.

Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear.Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury.Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes.Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time.

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.

Fyn-binding protein ADAP supports actin organization in podocytes.

The renal podocyte is central to the filtration function of the kidney that is dependent on maintaining both highly organized, branched cell structures forming foot processes, and a unique cell-cell junction, the slit diaphragm. Our recent studies investigating the developmental formation of the slit diaphragm identified a novel claudin family tetraspannin, TM4SF10, which is a binding partner for ADAP (also known as Fyn binding protein Fyb). To investigate the role of ADAP in podocyte function in relation to Fyn and TM4SF10, we examined ADAP knockout (KO) mice and podocytes. ADAP KO mice developed glomerular pathology that began as hyalinosis and progressed to glomerulosclerosis, with aged male animals developing low levels of albuminuria. Podocyte cell lines established from the KO mice had slower attachment kinetics compared to wild-type cells, although this did not affect the total number of attached cells nor the ability to form focal contacts. After attachment, the ADAP KO cells did not attain typical podocyte morphology, lacking the elaborate cell protrusions typical of wild-type podocytes, with the actin cytoskeleton forming circumferential stress fibers. The absence of ADAP did not alter Fyn levels nor were there differences between KO and wild-type podocytes in the reduction of Fyn activating phosphorylation events with puromycin aminonucleoside treatment. In the setting of endogenous TM4SF10 overexpression, the absence of ADAP altered the formation of cell-cell contacts containing TM4SF10. These studies suggest ADAP does not alter Fyn activity in podocytes, but appears to mediate downstream effects of Fyn controlled by TM4SF10 involving actin cytoskeleton organization.

The Cell Biology of APOL1.

The association of variants in the APOL1 gene, which encodes apolipoprotein L1 (APOL1), with progressive nondiabetic kidney diseases in African Americans has prompted intense investigation into the function(s) of APOL1. APOL1 is an innate immune effector that protects human beings from infection by some trypanosomal parasites. We review the data characterizing APOL1 trypanolytic function, which has been a basis for studies of APOL1 function in mammalian cells. Subsequently, we discuss the studies that use animal models, mammalian cell culture models, and kidney biopsy tissue to discover the mechanisms of variant APOL1-associated kidney diseases.