Direct Action of Endothelin-1 on Podocytes Promotes Diabetic Glomerulosclerosis

The endothelin system has emerged as a novel target for the treatment of diabetic nephropathy. Endothelin-1 promotes mesangial cell proliferation and sclerosis. However, no direct pathogenic effect of endothelin-1 on podocytes has been shown in vivo and endothelin-1 signaling in podocytes has not been investigated. This study investigated endothelin effects in podocytes during experimental diabetic nephropathy. Stimulation of primary mouse podocytes with endothelin-1 elicited rapid calcium transients mediated by endothelin type A receptors (ETARs) and endothelin type B receptors (ETBRs). We then generated mice with a podocyte-specific double deletion of ETAR and ETBR (NPHS2-Cre×Ednra^lox/lox×Ednrb^lox/lox [Pod-ETRKO]). In vitro, treatment with endothelin-1 increased total β-catenin and phospho-NF-κB expression in wild-type glomeruli, but this effect was attenuated in Pod-ETRKO glomeruli. After streptozotocin injection to induce diabetes, wild-type mice developed mild diabetic nephropathy with microalbuminuria, mesangial matrix expansion, glomerular basement membrane thickening, and podocyte loss, whereas Pod-ETRKO mice presented less albuminuria and were completely protected from glomerulosclerosis and podocyte loss, even when uninephrectomized. Moreover, glomeruli from normal and diabetic Pod-ETRKO mice expressed substantially less total β-catenin and phospho-NF-κB compared with glomeruli from counterpart wild-type mice. This evidence suggests that endothelin-1 drives development of glomerulosclerosis and podocyte loss through direct activation of endothelin receptors and NF-κB and β-catenin pathways in podocytes. Notably, both the expression and function of the ETBR subtype were found to be important. Furthermore, these results indicate that activation of the endothelin-1 pathways selectively in podocytes mediates pathophysiologic crosstalk that influences mesangial architecture and sclerosis.Diabetic nephropathy (DN) is the major microvascular complication of diabetes and the leading cause of ESRD in industrialized countries.¹ Clinically, DN is manifested by microalbuminuria, proteinuria, and progressive glomerular dysfunction. The main pathologic features of DN include podocyte loss, mesangial cell hypertrophy, glomerular basement membrane thickening, glomerulosclerosis, and tubulointerstitial fibrosis.^2–4 With the current standard therapies, including angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers, only partial renal protection is obtained.^5–7 Thus, it is of particular importance to understand more about the pathogenesis of DN and to identify novel therapeutic targets in order to develop new therapies that will prevent or delay the progression of DN.The endothelin (ET) system has recently emerged as an interesting novel target for the treatment of DN. ET-1 is a powerful mitogen and vasoconstrictor that influences a wide variety of organ functions and has been implicated in several cardiovascular and renal pathologies.^8,9 ET-1 signals through two G protein–coupled receptors (GPCRs), endothelin receptor type A (ETAR) and endothelin receptor type B (ETBR), and can lead to the activation of a variety of signaling cascades such as NF-κB,¹⁰ β-catenin, phosphoinositide 3-kinase, or mitogen-activated protein kinase.^11–13 Both receptors are present in the kidney.^14–17 It was early recognized that ET-1 displays proliferative effects on mesangial cells that are mediated by ETAR.^18,19 At the glomerular level, ET-1 promotes mesangial cell proliferation, sclerosis, and podocyte injury, although it is not demonstrated whether this latter effect is direct.^8,18,20Several lines of evidence suggest a specific role for the ET signaling pathway in the pathogenesis of DN. ET-1 expression is increased in kidneys with DN and higher ET-1 concentrations are found in the circulation of patients with DN as well as in animal models of DN.^21–25 In diabetic db/db mice, mesangial matrix expansion was shown to be temporally and spatially associated with glomerular immunoreactivity for ET-1.²⁶ ET receptor (ETR) blockers have been shown to be nephroprotective in diabetic animals in a BP-independent manner.^27–32 In patients with DN, results from clinical trials of ETR blockers depend on the general cardiovascular status of patients modulating tolerance to sodium and water retention as well as on the drug used.^33,34 Nevertheless, recent clinical studies are encouraging and suggest that ETAR antagonists are not only capable of promoting a regression of proteinuria, but may also limit glomerulosclerosis-related renal injury.^31,32,35 ETAR antagonists were found to have anti-inflammatory and antifibrotic effects during experimental DN,^29,36 but the cell types that promote DN under the influence of ET-1 are still not known. Furthermore, despite the major role of podocyte dysfunction in DN, the specific involvement of ET-1 signaling in podocytes has not been fully investigated.⁸ Therefore, this study aimed to investigate the ET-1 signaling pathway in podocytes during diabetes-induced nephropathy.In this study, we show that mice with a podocyte-specific double deletion of Ednar and Ednbr alleles are protected from diabetes-induced glomerulosclerosis and podocyte loss. We found the first evidence that ET-1 activation in the kidney drives development of diabetic glomerulosclerosis and podocyte loss with direct activation of ETRs and NF-κB and β-catenin pathways in podocytes. Surprisingly, the ETBR subtype was found to be important both at the expression level and the functional level. Ednbr mRNA expression in primary podocytes was found to be two times higher than Ednar expression, and a ETB agonist elicited calcium mobilization with β-catenin and NF-κB signaling. Furthermore, these results indicate that selective activation of the ETR pathways in podocytes is involved in pathophysiologic cellular crosstalk that influences mesangial architecture and sclerosis.     

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.     

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.     

Role of Podocyte B7-1 in Diabetic Nephropathy

Podocyte injury and resulting albuminuria are hallmarks of diabetic nephropathy, but targeted therapies to halt or prevent these complications are currently not available. Here, we show that the immune-related molecule B7-1/CD80 is a critical mediator of podocyte injury in type 2 diabetic nephropathy. We report the induction of podocyte B7-1 in kidney biopsy specimens from patients with type 2 diabetes. Genetic and epidemiologic studies revealed the association of two single nucleotide polymorphisms at the B7-1 gene with diabetic nephropathy. Furthermore, increased levels of the soluble isoform of the B7-1 ligand CD28 correlated with the progression to ESRD in individuals with type 2 diabetes. In vitro, high glucose conditions prompted the phosphatidylinositol 3 kinase–dependent upregulation of B7-1 in podocytes, and the ectopic expression of B7-1 in podocytes increased apoptosis and induced disruption of the cytoskeleton that were reversed by the B7-1 inhibitor CTLA4-Ig. Podocyte expression of B7-1 was also induced in vivo in two murine models of diabetic nephropathy, and treatment with CTLA4-Ig prevented increased urinary albumin excretion and improved kidney pathology in these animals. Taken together, these results identify B7-1 inhibition as a potential therapeutic strategy for the prevention or treatment of diabetic nephropathy.Type 2 diabetes (T2D) is rapidly becoming the leading cause of ESRD.^1,2 Despite much progress and an overall improvement in the treatment of diabetic nephropathy (DN), the development of ESRD remains an epidemic problem.³ Podocyte foot processes, separated by narrow spaces, constitute the final barrier to urinary protein loss by creating the porous membrane slit diaphragm, the integrity of which is essential for retaining proteins during filtration.^4,5 A primary hallmark of DN is the progressive damage and death of glomerular podocytes,^1,6–9 resulting in the leaking of proteins into the urine.⁴B7-1 is an immune-related protein found on antigen-presenting cells that interacts with CD28 and CTLA4 on T cells, thus providing positive or negative costimulatory signals necessary for T-cell activation and survival.¹⁰ Induction of podocyte B7-1 is associated with development of proteinuria in human and murine lupus nephritis, in α₃ integrin knockout mice, in nephrin knockout mice, and in mice with LPS-induced proteinuria.⁵ The latter study also reported that podocytes exposed to LPS upregulate B7-1 in vitro and in vivo, thus leading to podocyte abnormalities and proteinuria.⁵ Of note, B7-1 knockout (B7-1^−/−) mice are protected from LPS-induced albuminuria, suggesting a causal link between podocyte B7-1 expression and proteinuria.⁵Abatacept (CTLA4-Ig) is an inhibitor of B7-1 that is currently used to treat autoimmune diseases.¹¹ Here we show that high glucose induces podocyte B7-1 expression, thereby contributing to podocyte morphologic alterations and ultimately to DN, and that B7-1 blockade with CTLA4-Ig can protect podocytes from high glucose–induced injuries. These results suggest that the CD28/B7-1 pathway is relevant to the pathogenesis of T2D DN in humans, and CTLA4-Ig treatment may therefore offer a therapeutic strategy to combat DN. This notion is further supported by our recent findings showing that Abatacept is a therapy for proteinuria in patients with podocyte B7-1–positive FSGS.¹²     

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.     

Association of 18 Confirmed Susceptibility Loci for Type 2 Diabetes With Indices of Insulin Release, Proinsulin Conversion, and Insulin Sensitivity in 5,327 Nondiabetic Finnish Men

OBJECTIVE

We investigated the effects of 18 confirmed type 2 diabetes risk single nucleotide polymorphisms (SNPs) on insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin.

RESEARCH DESIGN AND METHODS

A total of 5,327 nondiabetic men (age 58 ± 7 years, BMI 27.0 ± 3.8 kg/m²) from a large population-based cohort were included. Oral glucose tolerance tests and genotyping of SNPs in or near PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, LOC387761, CDKN2B, IGF2BP2, CDKAL1, HNF1B, WFS1, JAZF1, CDC123, TSPAN8, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B were performed. HNF1B rs757210 was excluded because of failure to achieve Hardy-Weinberg equilibrium.

RESULTS

Six SNPs (TCF7L2, SLC30A8, HHEX, CDKN2B, CDKAL1, and MTNR1B) were significantly (P < 6.9 × 10⁻⁴) and two SNPs (KCNJ11 and IGF2BP2) were nominally (P < 0.05) associated with early-phase insulin release (InsAUC_0–30/GluAUC_0–30), adjusted for age, BMI, and insulin sensitivity (Matsuda ISI). Combined effects of these eight SNPs reached −32% reduction in InsAUC_0–30/GluAUC_0–30 in carriers of ≥11 vs. ≤3 weighted risk alleles. Four SNPs (SLC30A8, HHEX, CDKAL1, and TCF7L2) were significantly or nominally associated with indexes of proinsulin conversion. Three SNPs (KCNJ11, HHEX, and TSPAN8) were nominally associated with Matsuda ISI (adjusted for age and BMI). The effect of HHEX on Matsuda ISI became significant after additional adjustment for InsAUC_0–30/GluAUC_0–30. Nine SNPs did not show any associations with examined traits.

CONCLUSIONS

Eight type 2 diabetes–related loci were significantly or nominally associated with impaired early-phase insulin release. Effects of SLC30A8, HHEX, CDKAL1, and TCF7L2 on insulin release could be partially explained by impaired proinsulin conversion. HHEX might influence both insulin release and insulin sensitivity.Impaired insulin secretion and insulin resistance, two main pathophysiological mechanisms leading to type 2 diabetes, have a significant genetic component (1). Recent studies have confirmed 20 genetic loci reproducibly associated with type 2 diabetes (2–13). Three were previously known (PPARG, KCNJ11, and TCF7L2), whereas 17 loci were recently discovered either by genome-wide association studies (SLC30A8, HHEX-IDE, LOC387761, CDKN2A/2B, IGF2BP2, CDKAL1, FTO, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B), or candidate gene approach (WFS1 and HNF1B). The mechanisms by which these genes contribute to the development of type 2 diabetes are not fully understood.PPARG is the only gene from the 20 confirmed loci previously associated with insulin sensitivity (14,15). Association with impaired β-cell function has been reported for 14 loci (KCNJ11, SLC30A8, HHEX-IDE, CDKN2A/2B, IGF2BP2, CDKAL1, TCF7L2, WFS1, HNF1B, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, KCNQ1, and MTNR1B) (6,12,13,16–38). Although associations of variants in HHEX (16–22), CDKAL1 (6,21–26), TCF7L2 (22,27–30), and MTNR1B (13,31,32) with impaired insulin secretion seem to be consistent across different studies, information concerning other genes is limited (12,18–25,27,33–38). The mechanisms by which variants in these genes affect insulin secretion are unknown. However, a few recent studies suggested that variants in TCF7L2 (22,39–42), SLC30A8 (22), CDKAL1 (22), and MTNR1B (31) might influence insulin secretion by affecting the conversion of proinsulin to insulin. Variants of FTO have been shown to confer risk for type 2 diabetes through their association with obesity (7,16) and therefore were not included in this study.Large population-based studies can help to elucidate the underlying mechanisms by which single nucleotide polymorphisms (SNPs) of different risk genes predispose to type 2 diabetes. Therefore, we investigated confirmed type 2 diabetes–related loci for their associations with insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin in a population-based sample of 5,327 nondiabetic Finnish men.     

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.     

Identification of De Novo Synthesized and Relatively Older Proteins: Accelerated Oxidative Damage to De Novo Synthesized Apolipoprotein A-1 in Type 1 Diabetes

OBJECTIVE

The accumulation of old and damaged proteins likely contributes to complications of diabetes, but currently no methodology is available to measure the relative age of a specific protein alongside assessment of posttranslational modifications (PTM). To accomplish our goal of studying the impact of insulin deficiency and hyperglycemia in type 1 diabetes upon accumulation of old damaged isoforms of plasma apolipoprotein A-1 (ApoA-1), we sought to develop a novel methodology, which is reported here and can also be applied to other specific proteins.

RESEARCH DESIGN AND METHODS

To label newly synthesized proteins, [ring-¹³C₆]phenylalanine was intravenously infused for 8 h in type 1 diabetic participants (n = 7) during both insulin treatment and 8 h of insulin deprivation and in nondiabetic participants (n = 7). ApoA-1 isoforms were purified by two-dimensional gel electrophoresis (2DGE) and assessment of protein identity, PTM, and [ring-¹³C₆]phenylalanine isotopic enrichment (IE) was performed by tandem mass spectrometry.

RESULTS

Five isoforms of plasma ApoA-1 were identified by 2DGE including ApoA-1 precursor (pro-ApoA-1) that contained the relatively highest IE, whereas the older forms contained higher degrees of damage (carbonylation, deamidation) and far less IE. In type 1 diabetes, the relative ratio of IE of [ring-¹³C₆]phenylalanine in an older isoform versus pro-ApoA-1 was higher during insulin deprivation, indicating that de novo synthesized pro-ApoA-1 more rapidly accumulated damage, converting to mature ApoA-1.

CONCLUSIONS

We developed a mass spectrometry–based methodology to identify the relative age of protein isoforms. The results demonstrated accelerated oxidative damage to plasma ApoA-1, thus offering a potential mechanism underlying the impact of poor glycemic control in type 1 diabetic patients that affects a patient''s risk for vascular disease.There is substantial evidence to indicate that oxidative stress and subsequent oxidative damage is a major factor in the pathogenesis of diabetic complications (1,2). Oxidative damage of plasma proteins has been reported in patients with type 1 diabetes (3–5), and the accumulation of these damaged proteins is associated with many chronic complications of type 1 diabetes (4,6,7). Each protein does not exist as a homogenous pool within a tissue, but rather exists as a heterogeneous mix of isoforms of the same protein at different ages for which different amounts of time have passed following translation. A key determinant of the composition of the proteome is the rate at which the proteins are synthesized and removed (degraded) from a tissue, and this turnover, which replaces aged, damaged proteins with de novo synthesized proteins, is likely to maintain a relatively healthy composition of the proteome. A relative increase in isoforms of oxidatively damaged proteins can occur because of accelerated oxidative damage if these damaged proteins are not removed by degradation and replaced by de novo synthesized proteins. A better understanding of the oxidative damage to proteins, and their accumulation in proteins such as lipoproteins, is critical to further understand the pathophysiology of diabetic complications, especially since atherosclerosis and cardiovascular complications are common in people with diabetes. However, there has been no methodology to measure the relative age of proteins, and thus, hypotheses related to the accumulation of aged proteins could not yet be tested.In two-dimensional gel electrophoresis (2DGE) of tissues or body fluids, numerous protein gel spots can be identified, each representing the same protein identity (8–19). Often these numerous spots exist as charge variants and thus present as a train of spots horizontally adjacent to one another on the gel (8–19), each with similar molecular weights but with different isoelectric points (pI). This phenomenon has been observed for various proteins that have known relevance to disease, such as prostate-specific antigen (PSA) (18), heat shock proteins (15), fibrinogen (14), apolipoprotein A-1 (ApoA-1) (9–14), and many others. We hypothesized that the different protein isoforms in spot trains are related to different ages of proteins, and we hoped that experiments to further understand the nature of these proteins would improve the understanding of diseases that are related to an accumulation of aged proteins. Based on principles of tracer incorporation into proteins, it is reasonable to assume that during an intravenous infusion of a tracer such as [ring-¹³C₆]phenylalanine, both tracer and tracee will be incorporated into proteins synthesized during the course of the infusion. However, no [ring-¹³C₆]phenylalanine would be incorporated into proteins that were synthesized before the infusion, such as hours, days, or even years earlier. Thus, the young de novo synthesized proteins that were most recently translated will have relatively higher amounts of isotopic label than the protein isoforms that have primarily accumulated from the past. It is likely that high levels of oxidative damage and other modifications would be detected in older protein isoforms because they are exposed to the environmental stresses over longer periods of time.We studied insulin deprivation and treatment in type 1 diabetes, as it is known that insulin deprivation and subsequent hyperglycemia in type 1 diabetes leads to oxidative stress and an increased accumulation of oxidized plasma proteins (3–5). We focused our investigation on ApoA-1, which is a key protein component of HDL and is involved in cholesterol clearance from arteries. Considering preliminary knowledge of ApoA-1 isoforms (10,11), the importance of ApoA-1 in vascular health, the increased risk of macrovascular disease in type 1 diabetic patients (20), and our experience with resolving its isoforms on 2DGE, we chose ApoA-1 for the current study. We hypothesized that withdrawal of insulin treatment in type 1 diabetes would result in increased oxidative damage to the newly synthesized proteins and that these proteins with damage-induced charge alteration would be found at the locations in 2DGE where the older proteins are typically found.     

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.     

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

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

Targeted Glomerular Angiopoietin-1 Therapy for Early Diabetic Kidney Disease

Vascular growth factors play an important role in maintaining the structure and integrity of the glomerular filtration barrier. In healthy adult glomeruli, the proendothelial survival factors vascular endothelial growth factor-A (VEGF-A) and angiopoietin-1 are constitutively expressed in glomerular podocyte epithelia. We demonstrate that this milieu of vascular growth factors is altered in streptozotocin-induced type 1 diabetic mice, with decreased angiopoietin-1 levels, VEGF-A upregulation, decreased soluble VEGF receptor-1 (VEGFR1), and increased VEGFR2 phosphorylation. This was accompanied by marked albuminuria, nephromegaly, hyperfiltration, glomerular ultrastructural alterations, and aberrant angiogenesis. We subsequently hypothesized that restoration of angiopoietin-1 expression within glomeruli might ameliorate manifestations of early diabetic glomerulopathy. Podocyte-specific inducible repletion of angiopoietin-1 in diabetic mice caused a 70% reduction of albuminuria and prevented diabetes-induced glomerular endothelial cell proliferation; hyperfiltration and renal morphology were unchanged. Furthermore, angiopoietin-1 repletion in diabetic mice increased Tie-2 phosphorylation, elevated soluble VEGFR1, and was paralleled by a decrease in VEGFR2 phosphorylation and increased endothelial nitric oxide synthase Ser¹¹⁷⁷ phosphorylation. Diabetes-induced nephrin phosphorylation was also reduced in mice with angiopoietin-1 repletion. In conclusion, targeted angiopoietin-1 therapy shows promise as a renoprotective tool in the early stages of diabetic kidney disease.Diabetic nephropathy (DN) is the leading cause of ESRD characterized by structural changes in the kidney glomerular filtration barrier, including podocyte foot process fusion, basement membrane thickening, mesangial expansion, and abnormal angiogenesis leading to albuminuria.^1,2Healthy adult podocytes express vascular endothelial growth factor-A (VEGF-A) and angiopoietin-1 (Angpt1).^3,4 VEGF-A binds to its receptors VEGFR1 and VEGFR2; the former also exists as a soluble form (sVEGFR1), which is an inhibitor of VEGF-A signaling.⁵ Angpt1 phosphorylates the TEK tyrosine kinase (Tie-2) receptor.⁵ VEGFR2 and Tie-2 are mainly expressed by glomerular endothelia, and their concurrent activation is predicted to lead to endothelial survival and vessel stabilization.^3,5,6 In otherwise healthy animals, podocyte VEGF-A depletion leads to proteinuria, whereas downregulation of podocyte Angpt1 does not appear to compromise glomerular biology.^7,8 Several reports have suggested that DN kidneys have altered expression of VEGF-A, Angpt1, and also Angpt2, the natural antagonist of Angpt1.^8–10 In diabetic mice, deletion of podocyte Angpt1,⁸ or further overexpression of VEGF-A in podocytes, worsens glomerular morphology and enhances proteinuria.¹¹ Viral delivery of COMP-Angpt1¹² or either glomerular transgenic or systemic overexpression of sVEGFR1 ameliorates albuminuria in DN.^13,14Here we demonstrate an Angpt1 deficiency and enhanced VEGF-A signaling in diabetic mouse kidneys. This growth factor milieu is predicted to destabilize endothelia and enhance angiogenesis,⁵ an early feature of diabetic glomerulopathy.² Accordingly, we hypothesized that site-directed Angpt1 therapy would ameliorate early diabetic glomerulopathy; to test this we used an inducible podocyte-specific transgenic Angpt1 strategy.     

Urine Podocyte mRNAs,Proteinuria, and Progression in Human Glomerular Diseases

Model systems demonstrate that progression to ESRD is driven by progressive podocyte depletion (the podocyte depletion hypothesis) and can be noninvasively monitored through measurement of urine pellet podocyte mRNAs. To test these concepts in humans, we analyzed urine pellet mRNAs from 358 adult and pediatric kidney clinic patients and 291 controls (n=1143 samples). Compared with controls, urine podocyte mRNAs increased 79-fold (P<0.001) in patients with biopsy-proven glomerular disease and a 50% decrease in kidney function or progression to ESRD. An independent cohort of patients with Alport syndrome had a 23-fold increase in urinary podocyte mRNAs (P<0.001 compared with controls). Urinary podocyte mRNAs increased during active disease but returned to baseline on disease remission. Furthermore, urine podocyte mRNAs increased in all categories of glomerular disease evaluated, but levels ranged from high to normal, consistent with individual patient variability in the risk for progression. In contrast, urine podocyte mRNAs did not increase in polycystic kidney disease. The association between proteinuria and podocyturia varied markedly by glomerular disease type: a high correlation in minimal-change disease and a low correlation in membranous nephropathy. These data support the podocyte depletion hypothesis as the mechanism driving progression in all human glomerular diseases, suggest that urine pellet podocyte mRNAs could be useful for monitoring risk for progression and response to treatment, and provide novel insights into glomerular disease pathophysiology.Human glomerular diseases are heterogeneous but can collectively be viewed as a spectrum of podocytopathies.¹ Podocyte depletion per se causes glomerulosclerosis in model systems^2–5 and is associated with progressive glomerulosclerosis in humans.^6–12 Podocyte damage causes damage to other podocytes¹³ and activation of the renin-angiotensin system (RAS), which, in turn, drives angiotensin II–dependent further podocyte detachment from destabilized glomeruli.¹⁴ In model systems glomerulosclerosis is initiated when >30% of podocytes have been lost from glomeruli.¹⁴ Quantitative podocyte depletion from glomeruli results in mesangial expansion (10%–20% depletion), adhesions to the Bowman capsule (30% depletion), FSGS (30%–50% depletion), and then global sclerosis (70%–90% depletion) associated with interstitial fibrosis.^4,14 Model systems demonstrate that throughout the progression process podocytes continue to detach and appear in the urine, where they can be monitored noninvasively through quantitation of urine pellet mRNAs.^14–17 Collectively, these findings describe key elements of the podocyte depletion hypothesis for progression of glomerular diseases, whereby progressive depletion of podocytes leads through a series of stages to glomerulosclerosis and ultimately to ESRD.Although increased urine podocyte excretion has also been documented in some inflammatory and noninflammatory glomerular diseases in humans,^12,18–35 the role of podocyte depletion in progression remains unproven. We therefore used urine pellet mRNAs to test the hypothesis that progression to ESRD in human glomerular diseases would also be associated with increased urine podocyte mRNAs, regardless of the underlying cause of glomerular injury. Because proteinuria is a well established marker of kidney disease progression^36,37 but varies in extent and relationship to progression in different diseases, we also examined the relationship between proteinuria and rate of podocyte detachment. Establishing that human glomerular diseases follow the rules defined in model systems of progression would provide a foundation for understanding the progression mechanism in human glomerular diseases and the potential for urine podocyte mRNAs to be clinically useful.