Nck Proteins Maintain the Adult Glomerular Filtration Barrier

Within the glomerulus, the scaffolding protein nephrin bridges the actin-rich foot processes that extend from adjacent podocytes to form the slit diaphragm. Mutations affecting a number of slit diaphragm proteins, including nephrin, cause glomerular disease through rearrangement of the actin cytoskeleton and disruption of the filtration barrier. We recently established that the Nck family of Src homology 2 (SH2)/SH3 cytoskeletal adaptor proteins can mediate nephrin-dependent actin reorganization. Formation of foot processes requires expression of Nck in developing podocytes, but it is unknown whether Nck maintains podocyte structure and function throughout life. Here, we used an inducible transgenic strategy to delete Nck expression in adult mouse podocytes and found that loss of Nck expression rapidly led to proteinuria, glomerulosclerosis, and altered morphology of foot processes. We also found that podocyte injury reduced phosphorylation of nephrin in adult kidneys. These data suggest that Nck is required to maintain adult podocytes and that phosphotyrosine-based interactions with nephrin may occur in foot processes of resting, mature podocytes.Podocytes are unique epithelial cells within the kidney glomerulus that comprise the outermost layer of the blood filtration barrier.¹ Upon differentiation, podocytes extend numerous actin-based foot processes from their cell bodies that interdigitate and surround the glomerular capillary wall. At the interface of adjacent foot processes, a specialized intercellular junction known as the slit diaphragm is formed. The slit diaphragm apparently contributes to the morphology of foot processes through physical connection to the underlying actin cytoskeleton, as mutations affecting numerous slit diaphragm-associated proteins lead to actin rearrangement and foot process effacement.² In addition to genetic alterations, injury to podocytes as a consequence of diseases such as diabetes and hypertension as well as inflammation may also result in impaired slit diaphragm filtration leading to loss of protein in the urine (proteinuria) and subsequent renal failure.³Nephrin, encoded by NPHS1, is a transmembrane protein of the Ig superfamily that forms a key structural component of the slit diaphragm. Nephrin also functions as an intracellular signaling scaffold to recruit proteins such as podocin and CD2AP to the podocyte membrane.⁴ Upon clustering of adjacent nephrin molecules, the intracellular domain of nephrin becomes tyrosine phosphorylated by Src family kinases.^5,6 We and others have recently demonstrated that multiple phosphorylated tyrosine residues on nephrin can directly bind the Nck adaptor proteins.^7–10 Nck is a subfamily of two highly related proteins, Nck1 (also Nckα) and Nck2 (also Nckβ or Grb4), which are composed of three Src homology 3 (SH3) domains followed by a carboxy-terminal SH2 domain.^11,12 The Nck SH2 domain binds optimally to phosphorylated YDxV motifs on nephrin, whereas the SH3 domains interact with a wide array of effector proteins such as N-WASp that regulate cytoskeletal dynamics.¹³ Accordingly, recruitment of Nck to phosphorylated nephrin induces localized actin polymerization.^7–10Through conditional deletion of Nck2 in podocytes of Nck1-null embryonic mice, we have previously shown that expression of Nck in developing mouse podocytes is required for the formation of foot processes.⁷ These mice present with congenital nephrotic syndrome, similar to that observed in mice deficient in nephrin,^14,15 suggesting that Nck proteins might provide a link between nephrin and the actin cytoskeleton during the development of podocytes. While it is clear that Nck signaling is important in developing podocytes, whether it has a similar role in maintaining podocyte structure and function throughout life has remained less apparent. To address this, we have now investigated the role of Nck proteins in the maintenance of podocyte foot processes in the adult kidney. Using a strategy that allows deletion of Nck2 expression in a conditional and inducible fashion, we found that Nck proteins are also required in the adult glomerulus to maintain proper podocyte morphology. We have also developed a series of antibodies that specifically recognize individual phosphorylated nephrin YDxV sites, which we have used to confirm nephrin tyrosine phosphorylation in adult kidneys. Together these results support an emerging hypothesis that nephrin phosphorylation is retained in differentiated podocytes and that connection to the actin cytoskeleton via Nck may be an ongoing requirement to maintain the functional integrity of the slit diaphragm.     

Slit Diaphragms Contain Tight Junction Proteins

Slit diaphragms are essential components of the glomerular filtration apparatus, as changes in these junctions are the hallmark of proteinuric diseases. Slit diaphragms, considered specialized adherens junctions, contain both unique membrane proteins (e.g., nephrin, podocin, and Neph1) and typical adherens junction proteins (e.g., P-cadherin, FAT, and catenins). Whether slit diaphragms also contain tight junction proteins is unknown. Here, immunofluorescence, immunogold labeling, and cell fractionation demonstrated that rat slit diaphragms contain the tight junction proteins JAM-A (junctional adhesion molecule A), occludin, and cingulin. We found these proteins in the same protein complexes as nephrin, podocin, CD2AP, ZO-1, and Neph1 by cosedimentation, coimmunoprecipitation, and pull-down assays. PAN nephrosis increased the protein levels of JAM-A, occludin, cingulin, and ZO-1 several-fold in glomeruli and loosened their attachment to the actin cytoskeleton. These data extend current information about the molecular composition of slit diaphragms by demonstrating the presence of tight junction proteins, although slit diaphragms lack the characteristic morphologic features of tight junctions. The contribution of these proteins to the assembly of slit diaphragms and potential signaling cascades requires further investigation.Slit diaphragms are specialized cell-cell junctions located between mature podocytes that have fascinated cell biologists and nephrologists for more than 40 yr.¹ In contrast to podocytes, most other epithelial cells have junctional complexes composed of tight junctions and adherens junctions. Slit diaphragms originate from typical apical junctional complexes between primordial epithelia of the early S-shaped body. These junctional complexes migrate in a zipper-like fashion to the base of the cell where tight junctions persist as interdigitation of the foot processes begins.^2,3 Slit diaphragms appear during the capillary loop stage and gradually replace tight junctions. In many diseases associated with proteinuria and foot process loss or effacement, there is a rerun in reverse of this developmental sequence, and tight junctions reappear between adjoining foot processes.^4–6Major progress has been made recently in establishing the molecular make-up of the slit diaphragms. Several integral membrane proteins, including nephrin,⁷ podocin,⁸ and Neph1,⁹ not found in other junctions, have been identified as slit diaphragm components. Slit diaphragms are currently looked upon as signaling platforms in which nephrin and Neph1 transduce major signals that serve to maintain the filtration slits and to regulate podocyte shape through interaction of slit diaphragm proteins with the actin cytoskeleton.¹⁰ Mutations in nephrin,⁷ Neph1,⁹ and podocin⁸ have been linked to diseases associated with foot process effacement and proteinuria. In addition to these specialized slit diaphragm proteins, a number of other proteins that are associated with junctions in other locations are also concentrated at the slit diaphragms, including the adherens junction proteins P-cadherin,¹¹ FAT,¹² β-catenin,¹¹ and p120 catenin;¹³ scaffold proteins such as ZO-1,^14,15 CD2AP,¹⁶ MAGI-2,¹⁷ and CASK;¹³ and actin binding proteins, including IQGAP¹⁷ and α-actinin 4.^17,18 Because slit diaphragms share some morphologic features with adherens junctions and contain P-cadherin and catenins, slit diaphragms are assumed to represent modified adherens junctions.¹¹ However, several scaffold proteins that are often associated with tight junctions (i.e., ZO-1,^14,15 MAGI-1,¹⁹ MAGI-2,¹⁷ and CASK¹³) are present at slit diaphragms and have been shown to associate with nephrin. Based on their derivation from typical tight junctions² and the fact that they are replaced by tight junctions in nephrosis,^4,6 we reasoned that slit diaphragms might also contain membrane proteins normally associated with tight junctions.In this paper, we used morphological, biochemical, and bioinformatics techniques to investigate the expression of representative tight junction proteins in glomeruli in situ and in slit diaphragm-enriched fractions. Here, we document the presence of several tight junction proteins in slit diaphragms and demonstrate their interactions with slit diaphragm proteins in both normal and PAN nephrotic rats. The presence of tight junction proteins in slit diaphragms adds a new dimension to understanding the organization and functions of these junctions.     

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry–based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.The kidney filter consists of three layers: fenestrated endothelial cells, the glomerular basement membrane, and podocytes.¹ Damage to any of these compartments becomes clinically evident as proteinuria and the development of kidney disease.² Of particular importance for the regulation of podocyte biology through signaling is the slit diaphragm, a specialized intercellular junction that bridges the 40-nm gap in between foot processes of neighboring podocytes. It also serves as a signaling platform regulating podocyte function. Mutations in genes encoding for components of the slit diaphragm, such as nephrin,³ podocin,⁴ CD2AP,⁵ and TRPC6,^6,7 are important causes of genetic forms of proteinuria. Alteration of these proteins results in defective signaling causing podocyte dysfunction, progressive glomerulosclerosis, and kidney failure. The slit diaphragm protein complex is a lipid-multiprotein supercomplex.⁸ Of central importance to the integrity and function of the protein complex is the prohibitin homology (PHB) domain protein podocin,⁹ which forms multimeric complexes and is required to control signal transduction through associated transmembrane proteins.^10,11Signaling processes governing podocyte function, integrity, and survival largely depend on signaling processes involving phosphorylation.^12,13 Comprehensive analyses of the signaling events in podocytes in vivo have been hampered by the fact that interference with these signaling cascades by genetic deletion often results in massively disrupted and dysfunctional podocytes. One of the primary aims of this study was to use phosphoproteomics to analyze thousands of phosphorylation sites in native murine glomeruli within single samples. Within this study, we show that this approach allows the introduction of new concepts into signaling processes at the kidney filtration barrier.     

Inducible overexpression of sFlt-1 in podocytes ameliorates glomerulopathy in diabetic mice

OBJECTIVE—Podocyte-specific, doxycycline (DOX)-inducible overexpression of soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) in adult mice was used to investigate the role of the VEGF-A/VEGF receptor (VEGFR) system in diabetic glomerulopathy.RESEARCH DESIGN AND METHODS—We studied nondiabetic and diabetic transgenic mice and wild-type controls treated with vehicle (VEH) or DOX for 10 weeks. Glycemia was measured by a glucose-oxidase method and blood pressure by a noninvasive technique. sFlt-1, VEGF-A, VEGFR2, and nephrin protein expression in renal cortex were determined by Western immunoblotting; urine sFlt-1, urine free VEGF-A, and albuminuria by enzyme-linked immunosorbent assay; glomerular ultrastructure by electron microscopy; and VEGFR1 and VEGFR2 cellular localization with Immunogold techniques.RESULTS—Nondiabetic DOX-treated transgenic mice showed a twofold increase in cortex sFlt-1 expression and a fourfold increase in sFlt-1 urine excretion (P < 0.001). Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04). VEGFR2 expression was unchanged, whereas its activation was reduced in DOX-treated transgenic mice (P < 0.02). Albuminuria and glomerular morphology were similar among groups. DOX-treated transgenic diabetic mice showed a 60% increase in 24-h urine sFlt-1 excretion and an ∼70% decrease in urine free VEGF-A compared with VEH-treated diabetic mice (P < 0.04) and had lower urine albumin excretion at 10 weeks than VEH-treated diabetic (d) mice: d-VEH vs. d-DOX, geometric mean (95% CI), 117.5 (69–199) vs. 43 (26.8–69) μg/24 h (P = 0.003). Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-β1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05). Diabetes-induced VEGF-A and nephrin expression were not affected in DOX-treated mice.CONCLUSIONS—Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.Vascular endothelial growth factor (VEGF)-A is constitutively expressed in glomerular visceral cells (podocytes). Paracrine VEGF-A signaling occurs between podocytes and adjacent endothelial and mesangial cells, which express VEGF receptors (VEGFRs) 1 and 2 (1–3), and both autocrine and paracrine signaling may occur in podocytes themselves (4).VEGF-A has been implicated in the regulation of glomerular barrier properties to protein filtration. In normal animals (5,6) and in cancer patients (7), inhibition of VEGF-A results in proteinuria; while in proteinuric conditions, which are associated with glomerular VEGF-A upregulation such as diabetes, systemic inhibition of VEGF ameliorates albuminuria (8–10). This evidence suggests that a tight regulation of VEGF-A expression level is required to maintain the physiological permselective properties of the glomerular filter.The results of previous studies conducted using either VEGF gene targeting techniques (5) or by administration of inhibitory agents of the VEGF/VEGFR system such as antibodies (6,8,9) or chemicals (10) may have been affected by potential interference with animal organ development and lack of tissue specificity in the mechanisms of action of systemic inhibitors. Soluble VEGF receptor-1 (sFlt-1), a splice variant of the VEGFR1, lacks the transmembrane and complete intracellular tyrosine kinase domain of VEGFR1 but binds to VEGF with the same affinity and specificity as that of the full-length receptor (11,12) and has potent and selective VEGF inhibitory action (11). sFlt-1 acts in two major ways: it can sequester VEGF competing for its binding to the VEGF receptors or can form heterodimers with the extracellular region of the membrane spanning VEGFR1 and VEGFR2, thus inhibiting the activation of downstream signaling pathways (11,12).To target the action of the podocyte-expressed VEGF-A, we developed a transgenic mouse model to overexpress sFlt-1 specifically at the podocyte level with an inducible expression system that is induced only after complete development, in the adult animal, by the administration of doxycycline (Tet-on). The aim of this study was to investigate the role of VEGF-A upregulation in the pathogenesis of diabetic glomerulopathy by locally inhibiting podocyte-expressed VEGF-A activity.     

Recruitment of Podocytes from Glomerular Parietal Epithelial Cells

Loss of a critical number of podocytes from the glomerular tuft leads to glomerulosclerosis. Even in health, some podocytes are lost into the urine. Because podocytes themselves cannot regenerate, we postulated that glomerular parietal epithelial cells (PECs), which proliferate throughout life and adjoin podocytes, may migrate to the glomerular tuft and differentiate into podocytes. Here, we describe transitional cells at the glomerular vascular stalk that exhibit features of both PECs and podocytes. Metabolic labeling in juvenile rats suggested that PECs migrate to become podocytes. To prove this, we generated triple-transgenic mice that allowed specific and irreversible labeling of PECs upon administration of doxycycline. PECs were followed in juvenile mice beginning from either postnatal day 5 or after nephrogenesis had ceased at postnatal day 10. In both cases, the number of genetically labeled cells increased over time. All genetically labeled cells coexpressed podocyte marker proteins. In conclusion, we demonstrate for the first time recruitment of podocytes from PECs in juvenile mice. Unraveling the mechanisms of PEC recruitment onto the glomerular tuft may lead to novel therapeutic approaches to renal injury.Chronic kidney disease, resulting in renal failure and the need for lifelong renal replacement therapy, has become a significant problem worldwide. In the United States, approximately 7% of the total Medicare budget is spent on the treatment of ESRD, and projections suggest that the amount spent will increase by another 50% by 2020.¹Most renal pathologies that ultimately lead to ESRD originate within the glomerulus. It has now been established that a depletion of podocytes, the visceral epithelium of the capillary convolute (Figure 1), is central in this process. As soon as damage to the glomerular podocytes exceeds a certain threshold (approximately 30%), glomerulosclerosis ensues.² Indeed, in patients with a surgical reduction of ≥75% of renal mass, a relative lack of podocytes (podocytopenia) and subsequent FSGS in the originally healthy remnant kidney can lead to renal failure.³ Glomerulosclerosis is also the common final pathway of all glomerular diseases leading to ESRD.⁴ In glomerular diseases such as diabetic nephropathy, glomerulonephritides, or preeclampsia, significant numbers of podocytes are lost as a result of apoptosis, necrosis or excretion of living cells into the urine. Even in normal individuals, low numbers of living podocytes are continuously shed into the urine.^5–7 These numbers are too high to be compatible with renal survival for 80 yr, suggesting the existence of a regenerative mechanism. Also, the reversal of early glomerular damage in animal models and humans^8–10 argues for the existence of such a mechanism; however, podocytes are postmitotic cells that cannot undergo complete cell divisions and are therefore unable to regenerate themselves.^8–10 A potential mechanism for podocyte replacement from bone marrow–derived stem cells has been described in the Alport mouse model as well as in kidney transplants.^11–13 Nevertheless, most studies concluded that regeneration occurs predominantly from an as-yet-unknown source of resident renal cells.^12,14–16Open in a separate windowFigure 1.Renal glomerulus. The glomerular epithelium consists of PECs (red) and podocytes (Pod; blue), which reside on the capillary convolute. Both epithelia adjoin directly at the vascular pole (VP; arrow). At the tubular pole (TP), the parietal epithelium is connected to the epithelium of the proximal tubule. In male mice, this transition from PECs to proximal tubular cells often occurs within the glomerulus. The glomerular basement membrane (black) forms a continuous barrier between the glomerular epithelium and the endocapillary compartment that contains mesangial cells (shaded) and endothelial cells of the glomerular capillaries (*). Primary urine is filtered across the three-layered filtration barrier (endothelial cells, glomerular basement membrane, and Pod) into Bowman''s space (BS).In this study, we tested the hypothesis that glomerular parietal epithelial cells (PECs) lining the inner aspect of Bowman''s capsule migrate onto the glomerular tuft and differentiate into podocytes. Several arguments support this hypothesis. PECs are present in all species whose kidneys contain glomeruli. They are located within the same compartment and are in direct continuity with podocytes at the glomerular vascular stalk, so PECs do not have to cross an anatomic barrier such as the glomerular basement membrane, as was suggested for bone marrow–derived stem cells.^11–13 PECs proliferate lifelong at a relatively low frequency,¹⁷ express several stem cell marker proteins, and could be transdifferentiated in vitro into other cell types such as adipocytes or neuronal cells, suggesting that these cells retain multipotency.^9,18,19 In rodents, PECs do not express any known specific marker protein, which has so far precluded a detailed analysis of the function of these cells.In this work, we provide the first evidence that PECs possess the capability to migrate onto the glomerular tuft via the vascular stalk, where they differentiate into podocytes. This establishes that PECs represent an intrinsic cell population from which podocytes can be recruited.     

Depression,Pain Intensity,and Interference in Acute Spinal Cord Injury

Background:

The high prevalence of pain and depression in persons with spinal cord injury (SCI) is well known. However the link between pain intensity, interference, and depression, particularly in the acute period of injury, has not received sufficient attention in the literature.

Objective:

To investigate the relationship of depression, pain intensity, and pain interference in individuals undergoing acute inpatient rehabilitation for traumatic SCI.

Methods:

Participants completed a survey that included measures of depression (PHQ-9), pain intensity (“right now”), and pain interference (Brief Pain Inventory: general activity, mood, mobility, relations with others, sleep, and enjoyment of life). Demographic and injury characteristics and information about current use of antidepressants and pre-injury binge drinking also were collected. Hierarchical multiple regression was used to test depression models in 3 steps: (1) age, gender, days since injury, injury level, antidepressant use, and pre-injury binge drinking (controlling variables); (2) pain intensity; and (3) pain interference (each tested separately).

Results:

With one exception, pain interference was the only statistically significant independent variable in each of the final models. Although pain intensity accounted for only 0.2% to 1.2% of the depression variance, pain interference accounted for 13% to 26% of the variance in depression.

Conclusion:

Our results suggest that pain intensity alone is insufficient for understanding the relationship of pain and depression in acute SCI. Instead, the ways in which pain interferes with daily life appear to have a much greater bearing on depression than pain intensity alone in the acute setting.Key words: depression, pain, spinal cord injuriesThe high incidence and prevalence of pain following spinal cord injury (SCI) is well established^1–6 and associated with numerous poor health outcomes and low quality of life (QOL).^1,7,8 Although much of the literature on pain in SCI focuses on pain intensity, there is emerging interest in the role of pain interference or the extent to which pain interferes with daily activities of life.^7,9 With prevalence as high as 77% in SCI, pain interference impacts life activities such as exercise, sleep, work, and household chores.^2,7,10–13 Pain interference also has been associated with disease management self-efficacy in SCI.¹⁴ There is a significant relationship between pain intensity and interference in persons with SCI.⁷ Like pain, the high prevalence of depression after SCI is well-established.^15–17 Depression and pain often co-occur,^18,19 and their overlap ranges from 30% to 60%.¹⁹ Pain is also associated with greater duration of depressed mood.²⁰ Pain and depression share common biological pathways and neurotransmitter mechanisms,¹⁹ and pain has been shown to attenuate the response to depression treatment.^21,22Despite the interest in pain and depression after SCI and implications for the treatment of depression, their co-occurrence has received far less attention in the literature.²³ Greater pain has been associated with higher levels of depression in persons with SCI,^16,24 although this is not a consistent finding.²⁵ Similarly, depression in persons with SCI who also have pain appears to be worse than for persons with non-SCI pain, suggesting that the link between pain and depression may be more intense in the context of SCI.²⁶ In one of the few studies of pain intensity and depression in an acute SCI rehabilitation setting, Cairns et al ²⁷ found a co-occurrence of pain and depression in 22% to 35% of patients. This work also suggested an evolution of the relationship between pain and depression over the course of the inpatient stay, such that they become associated by discharge. Craig et al²⁸ found that pain levels at discharge from acute rehabilitation predicted depression at 2-year follow-up. Pain interference also has been associated with emotional functioning and QOL in persons with SCI^1,7,29,30 and appears to mediate the relationship between ambulation and depression.³¹Studies of pain and depression in person with SCI are often limited methodologically to examine the independent contributions of pain intensity and interference to depression in an acute setting. For example, they include only pain intensity^{16,23,25,28,30}; classify subjects by either pain plus depression²³ or pain versus no pain^8,28,30; use pain intensity and interference as predictor and outcome, respectively¹; collapse pain interference domains into a single score¹; or use only univariate tests (eg, correlations).^7,8,25,30 In addition, the vast majority focus on the chronic period of injury. To fill a gap in knowledge, we examined the independent contributions of pain intensity and pain interference to depression, while accounting for injury and demographic characteristics, antidepressant treatment, and pre-injury binge drinking in a sample of persons with acute SCI. We hypothesized that when accounting for both pain intensity and interference in the model, interference would have an independent and significant relationship with depression, above and beyond pain intensity.     

Phosphorylation of Nephrin Triggers Its Internalization by Raft-Mediated Endocytosis

Proper localization of nephrin determines integrity of the glomerular slit diaphragm. Slit diaphragm proteins assemble into functional signaling complexes on a raft-based platform, but how the trafficking of these proteins coordinates with their signaling function is unknown. Here, we demonstrate that a raft-mediated endocytic (RME) pathway internalizes nephrin. Nephrin internalization was slower with raft-mediated endocytosis than with classic clathrin-mediated endocytosis. Ultrastructurally, the RME pathway consisted of noncoated invaginations and was dependent on cholesterol and dynamin. Nephrin constituted a stable, signaling-competent microdomain through interaction with Fyn, a Src kinase, and podocin, a scaffold protein. Tyrosine phosphorylation of nephrin triggered its own RME-mediated internalization. Protamine-induced hyperphosphorylation of nephrin led to noncoated invaginations predominating over coated pits. These results demonstrate that an RME pathway couples nephrin internalization to its own signaling, suggesting that RME promotes proper spatiotemporal assembly of slit diaphragms during podocyte development or injury.Regulation of the leakiness and structural integrity of the slit diaphragm (SD) is a key process for maintenance of the glomerular filtration function.¹ Nephrin is a core structural component of the SD, and its ectodomains interact in a homophilic manner, thereby forming a zipper-like intercellular junction.^2,3 In addition to its structural component, nephrin transduces a phosphotyrosine signal through its cytoplasmic domain, which contains several phosphorylation sites for the Src-family kinases, such as Fyn. Ectodomain engagement of nephrin induces tyrosine phosphorylation via the Src-family kinases, which, in turn, leads to recruitment of an adaptor (e.g., Nck) and subsequent actin reorganization.^4,5SD proteins are organized within a specialized submicrodomain of the plasma membrane called rafts, liquid-ordered structures enriched in cholesterol and sphingolipids. They are therefore regarded as lipid–protein supercomplexes formed by a dynamic lateral assembly of the microdomains.⁶ Clustering of nephrin leads to sequential coalescence of preexisting small rafts, thereby forming a large-scale, stable signal platform through selective sequestration of adaptors and scaffolds.^7,8 In podocyte foot processes, a raft-resident scaffold protein, podocin, and a CD2-associated protein, CD2AP, facilitate the selective recruitment of signaling molecules and strengthen nephrin-based cell–cell attachment.^9–13 These scaffold proteins accommodate diverse functions of the SD complex: Cell survival, polarity, endocytosis, and cytoskeletal organization.⁶More than 70 nephrin mutations have been reported in familial nephrotic syndrome.^3,14,15 Several studies with humans and in animal nephrotic models showed that dysfunctional nephrin may be downregulated or mislocated in the effaced foot processes. These observations indicated that the spatiotemporal regulation of nephrin is a critical determinant of SD leakiness; however, the detailed mechanisms are still unclear. In this study, we focused on the role of endocytosis, a fundamental process that coordinates cell-surface expression and signal transduction.¹⁶ In mammalian cells, endocytosis is mediated via two principal routes: clathrin-mediated endocytosis (CME) and clathrin-independent, raft-mediated endocytosis (RME).^16–19 CME targets proteins to the early endosome, a sorting station directing vesicles to either recycling or degradation. Besides this “classic” CME pathway, RME has recently been the focus of intensive research, uncovering a new concept that the microdomain itself behaves as a vehicle for internalization. RME is generally defined by its clathrin independence, cholesterol sensitivity, and a typical morphology of smooth invaginations.^18,19 Several subtypes of RME pathways are differentially regulated by caveolin, dynamin, and a small GTPase, and they have been implicated in diverse biologic processes (e.g., viral infection, immunologic reactions, receptor signaling).¹⁹Podocytes can internalize circulating proteins as well as SD constituents.^20–22 In nephrotic podocytes, the number of endocytic vesicles is markedly increased and nephrin is downregulated and/or dislocated.^23,24 In view of accumulating evidence that SD proteins are assembled into functional signal complexes on a raft-based platform, we reasoned that an RME pathway could play a key role in spatiotemporal regulation of nephrin; however, it is still unclear how nephrin specifies CME or RME pathways and how these trafficking routes are coordinated with the signaling function. This study is the first to demonstrate that nephrin traffics via RME in cultured cells and podocytes in vivo. The results point to the possibility that this new RME pathway is implicated in remodeling and development of foot processes; these observations should attract further research focusing on disease pathogenesis and drug discovery.     

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.     

Podocyte Glutamatergic Signaling Contributes to the Function of the Glomerular Filtration Barrier

Podocytes possess the complete machinery for glutamatergic signaling, raising the possibility that neuron-like signaling contributes to glomerular function. To test this, we studied mice and cells lacking Rab3A, a small GTPase that regulates glutamate exocytosis. In addition, we blocked the glutamate ionotropic N-methyl-d-aspartate receptor (NMDAR) with specific antagonists. In mice, the absence of Rab3A and blockade of NMDAR both associated with an increased urinary albumin/creatinine ratio. In humans, NMDAR blockade, obtained by addition of ketamine to general anesthesia, also had an albuminuric effect. In vitro, Rab3A-null podocytes displayed a dysregulated release of glutamate with higher rates of spontaneous exocytosis, explained by a reduction in Rab3A effectors resulting in freedom of vesicles from the actin cytoskeleton. In addition, NMDAR antagonism led to profound cytoskeletal remodeling and redistribution of nephrin in cultured podocytes; the addition of the agonist NMDA reversed these changes. In summary, these results suggest that glutamatergic signaling driven by podocytes contributes to the integrity of the glomerular filtration barrier and that derangements in this signaling may lead to proteinuric renal diseases.It is widely recognized that most glomerular diseases are characterized by defects of the filtration barrier, where podocytes play a central role. Mutations of single podocyte proteins have been found at the basis of human nephrotic syndromes,¹ and podocyte deletion of the same molecules causes heavy proteinuria in experimental models.^2–8Podocytes are highly ramified cells: From the cell body depart a number of primary processes, further originating secondary foot processes. Starting from these features, it has been demonstrated that podocytes share numerous similarities with neurons: They both are terminally differentiated cells, have a common cytoskeletal organization, and have a common machinery of process formation.⁹ Furthermore, a number of expression-restricted proteins, such as nephrin,² Neph1 and Neph2,¹⁰ GLEPP1,¹¹ CAT3 and EAAT2,¹² synaptopodin,¹³ drebrin,¹⁴ and Sam68-like-MP2,¹⁵ specifically belong to the podocyte and the neuron.Our group has contributed to this line of research, initially by describing in podocytes the presence of Rab3A, a small GTPase that is mostly enriched in synaptic vesicles because it tightly modulates highly regulated exocytosis by acting through a number of effector molecules, including rabphilin-3a and Synapsin-I.¹⁶ After finding that in podocytes, as it occurs in neurons, Rab3A associates to glutamate-containing vesicles along cell processes, we discovered that podocytes are equipped with a complete neuron-like glutamatergic signaling system.¹⁷ We described that podocytes possess functional synaptic-like microvesicles and renal glomeruli express cognate glutamate transporters and receptors. These properties strengthened the analogies between podocytes and neurons and offered a rational interpretation to the biochemical similarity of foot process and synaptic adhesion complexes¹⁷; however, the role played by glutamate signaling in podocytes remained unanswered, and nothing was known about its relevance to podocyte and glomerular homeostasis. To get more details on the requirement of this neuron-like system of signaling by podocytes, we first conducted a preliminary analysis on its temporal appearance during podocyte differentiation. Then we studied conditions in which it was altered, on the vesicle and on the receptor side. The vesicular component was analyzed by studying the consequences of the absence of Rab3A. On the receptor side, we antagonized the ionotropic N-methyl-d-aspartate receptor (NMDAR), that we found present in human and rodent glomeruli, as well as in podocyte cell cultures.¹⁷ Both Rab3A and NMDAR1 glomerular synthesis were also confirmed by in situ hybridization (Supplemental Figure 1) and by microarray expression data.¹⁷     

Dietary Intake Relative to Cardiovascular Disease Risk Factors in Individuals With Chronic Spinal Cord Injury: A Pilot Study

Background:

The relationship between cardiovascular disease (CVD) risk factors and dietary intake is unknown among individuals with spinal cord injury (SCI).

Objective:

To investigate the relationship between consumption of selected food groups (dairy, whole grains, fruits, vegetables, and meat) and CVD risk factors in individuals with chronic SCI.

Methods:

A cross-sectional substudy of individuals with SCI to assess CVD risk factors and dietary intake in comparison with age-, gender-, and race-matched able-bodied individuals enrolled in the Coronary Artery Risk Development in Young Adults (CARDIA) study. Dietary history, blood pressure, waist circumference (WC), fasting blood glucose, high-sensitivity C-reactive protein (hs-CRP), lipids, glucose, and insulin data were collected from 100 SCI participants who were 38 to 55 years old with SCI >1 year and compared to 100 matched control participants from the CARDIA study.

Results:

Statistically significant differences between SCI and CARDIA participants were identified in WC (39.2 vs 36.2 in.; P < .001) and high-density lipoprotein cholesterol (HDL-C; 39.2 vs 47.5 mg/dL; P < .001). Blood pressure, total cholesterol, triglycerides, glucose, insulin, and hs-CRP were similar between SCI and CARDIA participants. No significant relation between CVD risk factors and selected food groups was seen in the SCI participants.

Conclusion:

SCI participants had adverse WC and HDL-C compared to controls. This study did not identify a relationship between consumption of selected food groups and CVD risk factors.Key words: cardiovascular disease risk factors, dietary intake, spinal cord injuryCardiovascular disease (CVD) is a leading cause of death in individuals with chronic spinal cord injuries (SCIs).^1–5 This is partly because SCI is associated with several metabolic CVD risk factors, including dyslipidemia,^6–10 glucose intolerance,^6,11–14 and diabetes.^15–17 In addition, persons with SCI exhibit elevated markers of inflammation^18,19 and endothelial activation²⁰ that are correlated with higher CVD prevalence.^21–23 Obesity, and specifically central obesity, another CVD risk factor,^24–26 is also common in this population.^12,27–29Dietary patterns with higher amounts of whole grains and fiber have been shown to improve lipid abnormalities,³⁰ glucose intolerance, diabetes mellitus,^31–34 hypertension,³⁵ and markers of inflammation³⁶ in the general population. These dietary patterns are also associated with lower levels of adiposity.³¹ Ludwig et al reported that the strong inverse associations between dietary fiber and multiple CVD risk factors – excessive weight gain, central adiposity, elevated blood pressure, hypertriglyceridemia, low high-density lipoprotein cholesterol (HDL-C), high low-density lipoprotein cholesterol (LDL-C), and high fibrinogen – were mediated, at least in part, by insulin levels.³⁷ Whole-grain food intake is also inversely associated with fasting insulin, insulin resistance, and the development of type 2 diabetes.^32,38,39Studies in the general population have also shown a positive association between the development of metabolic syndrome as well as heart disease and consumption of a Western diet, a diet characterized by high intake of processed and red meat and low intake of fruit, vegetables, whole grains, and dairy.^40,41 Red meat, which is high in saturated fat, has been shown to have an association with adverse levels of cholesterol and blood pressure and the development of obesity, metabolic syndrome, and diabetes.^40,42,43Numerous studies have shown that individuals with chronic SCI have poor diet quality.^44–49 A Canadian study found that only 26.7% of their sample was adherent to the recommendations about the consumption of fruit, vegetables, and grains from the “Eating Well with Canada’s Food Guide.”⁴⁴ Individuals with chronic SCI have also been found to have low fiber and high fat intakes when their diets were compared to dietary recommendations from the National Cholesterol Education Program,⁴⁶ the 2000 Dietary Guidelines for Americans,⁴⁹ and the recommended Dietary Reference Intakes and the Acceptable Macronutrient Distribution Range.^47,48However, unlike in the general population, the relationship between dietary intake and obesity and CVD risk factors is unknown in the chronic SCI population. If a dietary pattern consisting of higher intake of whole grains and dietary fiber is favorably associated with obesity and CVD risk factors in individuals with chronic SCI, then trials of increased intake of whole grains and fiber intake could be conducted to document health benefits and inform recommendations. The purpose of this pilot study is to investigate the association between selected food group intake and CVD risk factors in individuals with chronic SCI as compared to age-, gender-, and race-matched able-bodied individuals enrolled in the Coronary Artery Risk Development in Young Adults (CARDIA) study. Data will also be used to plan future studies in the relatively understudied field of CVD and nutrition in individuals with SCI.     

Predictors of Subclinical Atherosclerosis in Women With Spinal Cord Injury

Background:

Chronic spinal cord injury (SCI) is associated with an increase in risk factors for cardiovascular disease (CVD). In the general population, atherosclerosis in women occurs later than in men and usually presents differently. Associations between risk factors and incidence of CVD have not been studied in women with SCI.

Objective:

To determine which risk factors for CVD are associated with increased carotid intima-media thickness (CIMT), a common indicator of atherosclerosis, in women with SCI.

Methods:

One hundred and twenty-two females older than 18 years with traumatic SCI at least 2 years prior to entering the study were evaluated. Participants were asymptomatic and without evidence of CVD. Exclusion criteria were acute illness, overt heart disease, diabetes, and treatment with cardiac drugs, lipid-lowering medication, or antidiabetic agents. Measures for all participants were age, race, smoking status, level and completeness of injury, duration of injury, body mass index, serum lipids, fasting glucose, hemoglobin A1c, and ultrasonographic measurements of CIMT. Hierarchical multiple linear regression was conducted to predict CIMT from demographic and physiologic variables.

Results:

Several variables were significantly correlated with CIMT during univariate analyses, including glucose, hemoglobin A1c, age, and race/ethnicity; but only age was significant in the hierarchical regression analysis.

Conclusions:

Our data indicate the importance of CVD in women with SCI.Key words: age, cardiovascular disease, carotid intima-media thickness, hemoglobin A1c, risk factors, smokingThe secondary conditions of metabolic syndrome and cardiovascular disease (CVD) resulting from spinal cord injury (SCI ) are not well understood. In particular, persons with SCI have an increase in metabolic risk factors for cardiovascular disease (CVD),^1–5 but researchers have not determined whether this increase is associated with an increased incidence of CVD. The association has not been shown in reports on mortality or prevalence rates for CVD in people with SCI^6–12 or in the few studies that have appraised CVD in people with SCI using physiologic assessments.^13–18 Either the question was not addressed, or the evidence is insufficient due to low sample sizes and a lack of objective, prospective epidemiological studies assessing this question. Nevertheless, studies consistently show that metabolic syndrome is prevalent among individuals with SCI.^1–5,12 Metabolic syndrome consists of multiple interrelated risk factors that increase the risk for atherosclerotic heart disease by 1.5- to 3-fold.^19,20Compounding the uncertainty about the association of metabolic risk factors with CVD in SCI are possible gender differences.^21–24 Findings from studies of men with SCI might not apply to women with SCI. For example, the correlation between physical activity and high-density lipoprotein (HDL) levels in men with SCI is not found for women with SCI.^25,26 Furthermore, able-bodied women develop atherosclerosis later than do able-bodied men, and they usually present differently.²⁷ Some studies indicate that abnormal glucose metabolism may play a particularly important role in CVD in women²⁷; data from our group suggest that this is the case in women with SCI as well.¹⁵ Although women constitute 18% to 20% of the SCI population, no studies have evaluated cardiovascular health in women with chronic SCI.Carotid intima-media thickness (CIMT) is the most robust, highly tested, and often used noninvasive endpoint for assessing the progression of subclinical atherosclerosis in men and women of all ages.^28–46 For people with SCI, CIMT is a reliable surrogate measure of asymptomatic CVD.^15,47 The incidence of asymptomatic CVD appears to increase with the duration of SCI,¹⁵ where duration of injury is a cardiac risk factor independent of age.¹⁷ Moreover, CIMT is greater in men with SCI than in matched able-bodied controls,⁴⁸ indicating a subclinical and atypical presentation of CVD. A variety of studies have confirmed the usefulness of high-resolution B-mode ultrasound measurement of CIMT for quantitation of subclinical atherosclerosis.⁴⁹To better discern the association of risk factors with measures of subclinical atherosclerotic disease in women with SCI, we performed blood tests and ultrasonographic measurements of CIMT on 122 females with chronic SCI who were free of overt CVD. We tested for the 3 metabolic risk factors that are consistently identified in the varied definitions of metabolic syndrome: abnormal carbohydrate metabolism, abnormally high triglycerides, and abnormally low HDL cholesterol. We also tested for 4 other CVD risk factors: high levels of low-density lipoprotein (LDL), high total cholesterol, high body mass index (BMI), and a history of smoking.     

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.     

Adiponectin Promotes Functional Recovery after Podocyte Ablation

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

Distinct Roles for Basal and Induced COX-2 in Podocyte Injury

Transgenic mice that overexpress cyclooxygenase-2 (COX-2) selectively in podocytes are more susceptible to glomerular injury by adriamycin and puromycin (PAN). To investigate the potential roles of COX-2 metabolites, we studied mice with selective deletion of prostanoid receptors and generated conditionally immortalized podocyte lines from mice with either COX-2 deletion or overexpression. Podocytes that overexpressed COX-2 were virtually indistinguishable from wild-type podocytes but were significantly more sensitive to PAN-induced injury, produced more prostaglandin E₂ and thromboxane B₂, and had greater expression of prostaglandin E₂ receptor subtype 4 (EP₄) and thromboxane receptor (TP). Treatment of COX-2-overexpressing podocytes with a TP antagonist reduced apoptosis, but treatment with an EP₄ antagonist did not. In contrast, podocytes from COX-2-knockout mice exhibited increased apoptosis, markedly decreased cell adhesion, and prominent stress fibers. In vivo, selective deletion of podocyte EP₄ did not alter the increased sensitivity to adriamycin-induced injury observed in mice overexpressing podocyte COX-2. In contrast, genetic deletion of TP in these mice prevented adriamycin-induced injury, with attenuated albuminuria and foot process effacement. These results suggest that basal COX-2 may be important for podocyte survival, but overexpression of podocyte COX-2 increases susceptibility to podocyte injury, which is mediated, in part, by activation of the thromboxane receptor.Podocytes play a crucial role in regulation of glomerular function, and podocyte injury is an essential feature of progressive glomerular diseases. Although our understanding of podocyte biology has dramatically increased in recent years, mechanisms underlying functional and structural podocyte disturbances in renal diseases are still incompletely understood.¹ Recent studies indicate that local podocyte damage can spread to induce injury in otherwise healthy podocytes and further affect both glomerular endothelial and mesangial cells, implying that even limited podocyte injury might initiate a vicious cycle of progressive glomerular damage.²Mice with cyclooxygenase-2 (COX-2) gene deletion exhibit impaired glomerulogenesis and renal cortical development.³ However, increased expression of COX-2 in podocytes has been reported in various experimental models of progressive glomerular injury^4,5 and in cultured podocytes stimulated by mechanical stress.⁶ Furthermore, in models of renal ablation, diabetic nephropathy, and salt-sensitive hypertension, inhibition of COX-2 activity by selective COX-2 inhibitors significantly decreases proteinuria and progressive renal injury.^7–11 To determine if increased podocyte COX-2 expression plays a pathogenic role in glomerular injury, we recently generated COX-2 transgenic mice driven by a nephrin promoter, successfully inducing selective upregulation of COX-2 expression in podocytes. Although glomerular structure and function were normal at baseline in these transgenic mice, administration of either adriamycin or puromycin (PAN) led to significantly increased albuminuria compared with wild-type mice and induced further upregulation of COX-2 and downregulation of the slit diaphragm molecule nephrin. These studies suggested that increased podocyte COX-2 in response to injury may predispose the podocyte to further injury.^12,13 In cultured podocytes, mechanical stress induced COX-2 and expression of the prostaglandin E₂ (PGE₂) receptor subtype 4 (EP₄), and PGE₂ stimulation of stretched podocytes resulted in a loss of actin stress fiber organization.⁶ These results suggest that enhanced prostanoid signaling may be a facilitating event for morphologic changes and may directly influence podocyte function under pathophysiological conditions that promote synthesis of COX-2 metabolites. To investigate potential roles of COX-2 metabolites in podocyte function, we generated conditionally immortalized podocyte lines with either deficiency or overexpression of COX-2. In further in vitro and in vivo studies, we identified a role for COX-2-derived prostanoids as potential mediators of podocyte injury.     

Muscle Density and Bone Quality of the Distal Lower Extremity Among Individuals with Chronic Spinal Cord Injury

Background:

Understanding the related fates of muscle density and bone quality after chronic spinal cord injury (SCI) is an important initial step in determining endocrine-metabolic risk.

Objective:

To examine the associations between muscle density and indices of bone quality at the distal lower extremity of adults with chronic SCI.

Methods:

A secondary data analysis was conducted in 70 adults with chronic SCI (C2-T12; American Spinal Injury Association Impairment Scale [AIS] A-D; ≥2 years post injury). Muscle density and cross-sectional area (CSA) and bone quality indices (trabecular bone mineral density [TbBMD] at the distal tibia [4% site] and cortical thickness [CtTh], cortical area [CtAr], cortical BMD [CtBMD], and polar moment of inertia [PMI] at the tibial shaft [66% site]) were measured using peripheral quantitative computed tomography. Calf lower extremity motor score (cLEMS) was used as a clinical measure of muscle function. Multivariable linear regression analyses were performed to determine the strength of the muscle-bone associations after adjusting for confounding variables (sex, impairment severity [AIS A/B vs AIS C/D], duration of injury, and wheelchair use).

Results:

Muscle density was positively associated with TbBMD (b = 0.85 [0.04, 1.66]), CtTh (b = 0.02 [0.001, 0.034]), and CtBMD (b = 1.70 [0.71, 2.69]) (P < .05). Muscle CSA was most strongly associated with CtAr (b = 2.50 [0.12, 4.88]) and PMI (b = 731.8 [161.7, 1301.9]) (P < .05), whereas cLEMS was most strongly associated with TbBMD (b = 7.69 [4.63, 10.76]) (P < .001).

Conclusion:

Muscle density and function were most strongly associated with TbBMD at the distal tibia in adults with chronic SCI, whereas muscle size was most strongly associated with bone size and geometry at the tibial shaft.Key words: bone mineral density, bone quality, muscle density, muscle size, osteoporosis, peripheral quantitative computed tomography, spinal cord injurySpinal cord injury (SCI) is associated with sublesional muscle atrophy,^1–3 changes in muscle fiber type,^4,5 reductions in hip and knee region bone mineral density (BMD),^6–8 and increased central and regional adiposity after injury.^9,10 Adverse changes in muscle and bone health in individuals with SCI contribute to an increased risk of osteoporosis,^11–13 fragility fractures,¹⁴ and endocrine-metabolic disease (eg, diabetes, dyslipidemia, heart disease).^15–17 Crosssectional studies have shown a higher prevalence of lower extremity fragility fractures among individuals with SCI ranging from 1% to 34%.^18–20 Fragility fractures are associated with negative health and functional outcomes, including an increased risk of morbidity and hospitalization,^21,22 mobility limitations,²³ and a reduced quality of life.²⁴ Notably, individuals with SCI have a normal life expectancy, yet fracture rates increase annually from 1% per year in the first year to 4.6% per year in individuals greater than 20 years post injury.^25,26Muscle and bone are thought to function as a muscle-bone unit, wherein muscle contractions impose loading forces on bone that produce changes in bone geometry and structure.^27,28 A growing body of evidence has shown that individuals with SCI (predominantly those with motor complete injury) exhibit similar patterns of decline in muscle cross-sectional area (CSA) and BMD in the acute and subacute stages following injury.^4,11,29 Prospective studies have exhibited a decrease in BMD of 1.1% to 47% per year^6,7,30 and up to 73% in the 2 to 7 years following SCI.^8,14,31,32 Decreases in muscle CSA have been well-documented following SCI, with greater disuse atrophy observed after complete SCI versus incomplete SCI, presumably due to the absence of voluntary muscle contractions and associated mobility limitations.^1,2,16 Muscle quality is also compromised early after SCI, resulting in sublesional accumulation of adipose tissue in the chronic stage of injury^3,33,34; the exact time course of this event has been poorly elucidated to date. Adipose tissue deposition within and between skeletal muscle is linked to an increase in noncontractile muscle tissue and a reduction in muscle force-generating capacity on bone.^35,36 Skeletal muscle fat infiltration is up to 4 times more likely to occur in individuals with SCI,^1,16,37 contributing to metabolic complications (eg, glucose intolerance),¹⁶ reduced muscle strength and function,³⁸ and mobility limitations³ – all factors that may be associated with a deterioration in bone quality after SCI.The association between lean tissue mass and bone size (eg, BMD and bone mineral content) in individuals with SCI has been wellestablished using dual energy x-ray absorptiometry (DXA).^9,10,29,34 However, DXA is unable to measure true volumetric BMD (vBMD), bone geometry, and bone structure. Peripheral quantitative computed tomography (pQCT) is an imaging technique that improves our capacity to measure indices of bone quality and muscle density and CSA at fracture-prone sites (eg, tibia).^3,39 Recent evidence from cross-sectional pQCT studies has shown that muscle CSA and calf lower extremity motor score (cLEMS) were associated with indices of bone quality at the tibia in individuals with SCI.^13,40 However, neither study measured muscle density (a surrogate of fatty infiltration when evaluating the functional muscle-bone unit). Fatty infiltration of muscle is common after SCI^1,16,37 and may affect muscle function or the muscle-bone unit, but the association between muscle density and bone quality indices at the tibia in individuals with chronic SCI is unclear. Muscle density measured using pQCT may be an acceptable surrogate of muscle quality when it is difficult to assess muscle strength due to paralysis.^3,39 Additionally, investigating which muscle outcome (muscle density, CSA, cLEMS) is most strongly associated with vBMD and bone structure may inform modifiable targets for improving bone quality and reducing fracture risk after chronic SCI.The primary objective of this secondary analysis was to examine the associations between pQCTderived calf muscle density and trabecular vBMD at the tibia among adults with chronic SCI. The secondary objective was to examine the associations between calf muscle density, CSA, and function and tibial vBMD, cortical CSA and thickness, and polar moment of inertia (PMI). First, we hypothesize that calf muscle density will be a positive correlate of trabecular and cortical vBMD, cortical CSA and thickness, and PMI at the tibia in individuals with chronic SCI. Second, we hypothesize that of the key muscle variables (cLEMS, CSA and density), calf muscle density and cLEMS will be most strongly associated with trabecular vBMD, whereas calf muscle CSA will be most strongly associated with cortical CSA and PMI.     

Restoring Voluntary Grasping Function in Individuals with Incomplete Chronic Spinal Cord Injury: Pilot Study

Background:

Functional electrical stimulation (FES) therapy has been shown to be one of the most promising approaches for improving voluntary grasping function in individuals with subacute cervical spinal cord injury (SCI).

Objective:

To determine the effectiveness of FES therapy, as compared to conventional occupational therapy (COT), in improving voluntary hand function in individuals with chronic (≥24 months post injury), incomplete (American Spinal Injury Association Impairment Scale [AIS] B-D), C4 to C7 SCI.

Methods:

Eight participants were randomized to the intervention group (FES therapy; n = 5) or the control group (COT; n = 3). Both groups received 39 hours of therapy over 13 to 16 weeks. The primary outcome measure was the Toronto Rehabilitation Institute-Hand Function Test (TRI-HFT), and the secondary outcome measures were Graded Redefined Assessment of Strength Sensibility and Prehension (GRASSP), Functional Independence Measure (FIM) self-care subscore, and Spinal Cord Independence Measure (SCIM) self-care subscore. Outcome assessments were performed at baseline, after 39 sessions of therapy, and at 6 months following the baseline assessment.

Results:

After 39 sessions of therapy, the intervention group improved by 5.8 points on the TRI-HFT’s Object Manipulation Task, whereas the control group changed by only 1.17 points. Similarly, after 39 sessions of therapy, the intervention group improved by 4.6 points on the FIM self-care subscore, whereas the control group did not change at all.

Conclusion:

The results of the pilot data justify a clinical trial to compare FES therapy and COT alone to improve voluntary hand function in individuals with chronic incomplete tetraplegia.Key words: chronic patients, functional electrical stimulation, grasping, therapy, upper limbIn the United States and Canada, there is a steady rate of incidence and an increasing rate of prevalence of individuals living with spinal cord injury (SCI). For individuals with tetraplegia, hand function is essential for achieving a high level of independence in activities of daily living.^1–5 For the majority of individuals with tetraplegia, the recovery of hand function has been rated as their highest priority.⁵Traditionally, functional electrical stimulation (FES) has been used as a permanent neuroprosthesis to achieve this goal.^6–14 More recently, researchers have worked toward development of surface FES technologies that are meant to be used as shortterm therapies rather than permanent prosthesis. This therapy is frequently called FES therapy or FET. Most of the studies published to date, where FES therapy was used to help improve upper limb function, have been done in both the subacute and chronic stroke populations^15–23 and 2 have been done in the subacute SCI population.^1–3 With respect to the chronic SCI population, there are no studies to date that have looked at use of FES therapy for retraining upper limb function. In a review by Kloosterman et al,²⁴ the authors have discussed studies that have used various combinations of therapies for improving upper extremity function in chronic SCI individuals; however, the authors found that the only study that showed significant improvements before and after was the study published by Needham-Shropshire et al.²⁵ This study examined the effectiveness of neuromuscular stimulation (NMS)–assisted arm ergometry for strengthening triceps brachii. In this study, electrical stimulation was used to facilitate arm ergometry, and it was not used in the context of retraining reaching, grasping, and/or object manipulation.Since 2002, our team has been investigating whether FES therapy has the capacity to improve voluntary hand function in complete and incomplete subacute cervical SCI patients who are less than 180 days post injury at the time of recruitment in the study.^1–3 In randomized controlled trials (RCTs) conducted by our team, we found that FES therapy is able to restore voluntary reaching and grasping functions in individuals with subacute C4 to C7 incomplete SCI.^1–3 The changes observed were transformational; individuals who were unable to grasp at all were able to do so after only 40 one-hour sessions of the FES therapy, whereas the control group showed significantly less improvement. Inspired by these results, we decided to conduct a pilot RCT with chronic (≥24 months following injury) C4 to C7 SCI patients (American Spinal Injury Association Impairment Scale [AIS] B-D), which is presented in this article. The purpose of this pilot study was to determine whether the FES therapy is able to restore voluntary hand function in chronic tetraplegic individuals. Based on the results of our prior phase I¹ and phase II^2,3 RCTs in the subacute SCI population, we hypothesized that individuals with chronic tetraplegia who underwent the FES therapy (intervention group) may have greater improvements in voluntary hand function, especially in their ability to grasp and manipulate objects, and perform activities of daily living when compared to individuals who receive similar volume and duration of conventional occupational therapy (COT: control group).