Keeping the customers stratified: moving toward genetics-based treatment options in childhood NS.

In this issue, Bierzynska et al. report the results of analyzing a cohort of children with steroid-resistant nephrotic syndrome (SRNS) of childhood, correlating genetic studies with clinical outcomes. This approach has the potential to advance both our research into the causes and treatments of childhood SRNS and our ability to stratify patients into groups that are likely to be responsive to specific treatments. Here, we discuss some of the strengths and limitations of this report.

Enhancing clinical trial development for pediatric kidney diseases.

The conduct of clinical trials in small pediatric subspecialties such as pediatric nephrology is hampered by both clinical demands on the pediatric nephrologist and the small number of appropriate patients available for such studies. The American Society of Pediatric Nephrology Therapeutics Development Committee (TDC) was established to (1) identify the various stakeholders with interests and/or expertise related to clinical trials in children with kidney disease and (2) develop more effective partnerships among all parties regarding strategies for successful clinical trial development and execution. This article discusses the rationale, structure, and function of the TDC, the status of progress toward its goals, and the insights gained to date that may be useful for other subspecialties that face similar challenges.

Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3.

The function of hypoxia-inducible factor-1α (HIF-1α) in chronic kidney disease is disputed. Here we report that interactions of HIF-1α with transforming growth factor-ß (TGF-ß) signaling may promote its fibrotic effects. Knockout of HIF-1α is protective against glomerulosclerosis and glomerular type-I collagen accumulation in a mouse podocyte ablation model. Transcriptional analysis of cultured renal cells showed that α2(I) collagen expression is directly regulated by HIF-1α binding to a functional hypoxia-responsive element in its promoter at -335 relative to the transcription start site. Activation of COL1A2 transcription by HIF-1α occurred in the absence of hypoxia and is strongly enhanced by TGF-ß signaling. TGF-ß, in addition to increasing HIF-1α levels, increased both HIF-1α binding to the COL1A2 promoter and HIF-1α N-terminal transactivation domain activity. These effects of TGF-ß on HIF-1α were inhibited in Smad3-null mouse embryonic fibroblasts, suggesting a requirement for Smad3. Phosphorylated Smad3 also associated with the -335 hypoxia-responsive element of the COL1A2 promoter independent of a Smad DNA binding sequence. Smad3 binding to the -335 hypoxia-responsive element required HIF-1α both in vitro and in kidney lysate from the disease model, suggesting formation of an HIF-1α-Smad3 transcriptional complex. Thus, HIF-1α-Smad3 has a novel interaction in glomerulosclerosis.

TGFß receptor I transactivation mediates stretch-induced Pak1 activation and CTGF upregulation in mesangial cells.

Increased intraglomerular pressure is an important pathogenic determinant of kidney fibrosis in the progression of chronic kidney disease, and can be modeled by exposing glomerular mesangial cells (MC) to mechanical stretch. MC produce extracellular matrix and profibrotic cytokines, including connective tissue growth factor (CTGF) when stretched. We show that p21-activated kinase 1 (Pak1) is activated by stretch in MC in culture and in vivo in a process marked by elevated intraglomerular pressures. Its activation is essential for CTGF upregulation. Rac1 is an upstream regulator of Pak1 activation. Stretch induces transactivation of the type I transforming growth factor ß1 receptor (TßRI) independently of ligand binding. TßRI transactivation is required not only for Rac1/Pak1 activation, but also for activation of the canonical TGFß signaling intermediate Smad3. We show that Smad3 activation is an essential requirement for CTGF upregulation in MC under mechanical stress. Pak1 regulates Smad3 C-terminal phosphorylation and transcriptional activation. However, a second signaling pathway, that of RhoA/Rho-kinase and downstream Erk activation, is also required for stretch-induced CTGF upregulation in MC. Importantly, this is also regulated by Pak1. Thus, Pak1 serves as a novel central mediator in the stretch-induced upregulation of CTGF in MC.

Defining new surrogate markers for CKD progression.

At a recent meeting of international experts on clinical aspects of progressive chronic kidney disease (CKD), an extensive analysis of extant clinical trials was used to develop more effective and economical surrogate markers for CKD outcomes in adults. This article describes the reasons for this undertaking, the methods and conclusions of the meeting, and the relevance of these findings to pediatric nephrology.

Remnant nephron physiology and the progression of chronic kidney disease.

In chronic kidney disease, ongoing failure of individual nephrons leads to the progressive loss of renal function. This process results in part from a cellular and molecular response to injury that represents an attempt to maintain homeostasis but instead initiates a program that damages the nephron. As nephrons are lost, compensation by the remaining nephrons exacerbates glomerular pathophysiology. The delivery of excessive amounts of biologically active molecules to the distal nephron and tubulointerstitium generates inflammation and cellular dedifferentiation. Energy requirements of hyperfunctioning nephrons exceed the metabolic substrate available to the renal tubule, and inadequacy of the local vascular supply promotes hypoxia/ischemia and consequent acidosis and reactive oxygen species generation. In this way, mechanisms activated to maintain biological balance ultimately lead to demise of the nephron.

Phosphatidylinositol 3-kinase and Rab5 GTPase inversely regulate the Smad anchor for receptor activation (SARA) protein independently of transforming growth factor-ß1.

SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-ß1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-ß1 in that, unlike TGF-ß1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85α action, and co-immunoprecipitation of SARA and p85α confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either LY294002 or p85α knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via LY294002 or p85α knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle α-actin. Together, these data suggest that although TGF-ß1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K.

TGF-ß/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1α expression.

Transforming growth factor (TGF)-ß is a major mediator of kidney fibrosis. In the past decade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-ß-induced fibrogenesis. One such pathway involves mammalian target of rapamycin complex (mTORC)1. We recently reported that the hypoxia-inducible factor (HIF)-1 is essential for TGF-ß-induced collagen expression regardless of ambient oxygen tension. A modulator of HIF expression other than oxygen tension is mTORC1. We therefore sought to evaluate a possible role for mTORC1 activity in TGF-ß-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-ß in a TGF-ß receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-ß stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-ß-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-ß-stimulated increase in both hypoxia-responsive element (HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-ß-induced HRE activity. shRNA to Smad3 reduced HRE activity, while overexpression of Smad3 increased HIF-1α protein expression and activity in an mTORC1-dependent manner. Lastly, overexpression of HIF-1α bypassed the inhibitory effect of mTORC1 blockade on collagen expression. These results suggest that Smad3/mTORC1 interaction to promote HIF-1 expression is a key step in normoxic kidney fibrogenesis.

Hypoxia-inducible factor-2α and TGF-ß signaling interact to promote normoxic glomerular fibrogenesis.

Hypoxia-inducible factors (HIFs) are transcription factors consisting of an oxygen-sensitive α-subunit binding to a stable ß-subunit. HIFs regulate multiple signaling pathways that could contribute to fibrogenesis, supporting their potential role in hypoxia-mediated renal fibrosis. We previously reported that HIF-1 is upregulated and required for transforming growth factor (TGF)-ß induction of collagen in renal tubular cells. Here, we performed in vitro and in vivo studies of potential glomerular crosstalk between TGF-ß and normoxic HIF signaling. HIF-α has two major isoforms, HIF-1α and HIF-2α with different target gene sets. In cultured human mesangial cells, TGF-ß1 treatment increased both HIF-1α and HIF-2α expression in normoxia. TGF-ß1 did not increase HIF-1α/2α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances HIF-1α/2α expression through translation. TGF-ß receptor (ALK5) kinase activity was required for increased, TGF-ß-stimulated HIF-α expression in response to TGF-ß, and inhibiting PI3-kinase markedly decreased HIF-α expression. Blocking HIF-1α/2α expression using siRNA decreased basal and TGF-ß1-stimulated type I collagen expression, while overexpressing nondegradable HIF-α increased the collagen response, with HIF-2α being significantly more effective than HIF-1α. In adriamycin-induced mouse glomerulosclerosis, HIF-2α target genes were upregulated in sclerosing glomeruli. Taken together, our data demonstrate potential signaling interaction between TGF-ß and HIFs to promote renal fibrogenesis in normoxia and suggest that the HIF-2α isoform is more important during glomerulosclerosis.

Divergent roles of Smad3 and PI3-kinase in murine adriamycin nephropathy indicate distinct mechanisms of proteinuria and fibrogenesis.

Multiple transforming growth factor (TGF)-ß-induced fibrogenic signals have been described in vitro. To evaluate mechanisms in vivo, we used an adriamycin nephropathy model in 129x1/Svj mice that display massive proteinuria by days 5 to 7 and pathological findings similar to human focal segmental glomerulosclerosis by day 14. TGF-ß mRNA expression increased after day 7 along with nuclear translocation of the TGF-ß receptor-specific transcription factor Smad3. Inhibiting TGF-ß prevented both pathological changes and type-I collagen and fibronectin mRNA expression, but proteinuria persisted. Renal Akt was phosphorylated in adriamycin-treated mice, suggesting PI3-kinase activation. Expression of mRNA for the p110γ isozyme of PI3-kinase was specifically increased and p110γ colocalized with nephrin by immunohistochemistry early in disease. Nephrin levels subsequently decreased. Inhibition of p110γ by AS605240 preserved nephrin expression and prevented proteinuria. In cultured podocytes, adriamycin stimulated p110γ expression. AS605240, but not a TGF-ß receptor kinase inhibitor, prevented adriamycin-induced cytoskeletal disorganization and apoptosis, supporting a role for p110γ in podocyte injury. AS605240, at a dose that decreased proteinuria, prevented renal collagen mRNA expression in vivo but did not affect TGF-ß-stimulated collagen induction in vitro. Thus, PI3-kinase p110γ mediates initial podocyte injury and proteinuria, both of which precede TGF-ß-mediated glomerular scarring.

Tubulointerstitial injury and the progression of chronic kidney disease.

In chronic kidney disease (CKD), once injury from any number of disease processes reaches a threshold, there follows an apparently irreversible course toward decline in kidney function. The tubulointerstitium may play a key role in this common progression pathway. Direct injury, high metabolic demands, or stimuli from various other forms of renal dysfunction activate tubular cells. These, in turn, interact with interstitial tissue elements and inflammatory cells, causing further pathologic changes in the renal parenchyma. The tissue response to these changes thus generates a feed-forward loop of kidney injury and progressive loss of function. This article reviews the mechanisms of this negative cycle mediating CKD.

Clinical and translational science award T32/TL1 training programs: program goals and mentorship practices.

INTRODUCTION: A national survey characterized training and career development for translational researchers through Clinical and Translational Science Award (CTSA) T32/TL1 programs. This report summarizes program goals, trainee characteristics, and mentorship practices. METHODS: A web link to a voluntary survey was emailed to 51 active TL1 program directors and administrators. Descriptive analyses were performed on aggregate data. Qualitative data analysis used open coding of text followed by an axial coding strategy based on the grounded theory approach. RESULTS: Fifty out of 51 (98%) invited CTSA hubs responded. Training program goals were aligned with the CTSA mission. The trainee population consisted of predoctoral students (50%), postdoctoral fellows (30%), and health professional students in short-term (11%) or year-out (9%) research training. Forty percent of TL1 programs support both predoctoral and postdoctoral trainees. Trainees are diverse by academic affiliation, mostly from medicine, engineering, public health, non-health sciences, pharmacy, and nursing. Mentor training is offered by most programs, but mandatory at less than one-third of them. Most mentoring teams consist of two or more mentors. CONCLUSIONS: CTSA TL1 programs are distinct from other NIH-funded training programs in their focus on clinical and translational research, cross-disciplinary approaches, emphasis on team science, and integration of multiple trainee types. Trainees in nearly all TL1 programs were engaged in all phases of translational research (preclinical, clinical, implementation, public health), suggesting that the CTSA TL1 program is meeting the mandate of NCATS to provide training to develop the clinical and translational research workforce.

Loss of beta1-integrin enhances TGF-beta1-induced collagen expression in epithelial cells via increased alphavbeta3-integrin and Rac1 activity.

Transforming growth factor ß (TGF-ß) promotes tissue fibrosis via the receptor-specific Smad pathway and non-canonical pathways. We recently reported that TGF-ß1-stimulated collagen expression by cultured kidney cells requires integrin-dependent activation of focal adhesion kinase (FAK) and consequent ERK MAP kinase activity leading to Smad3 linker region phosphorylation. Here, we defined a role for αvß3-integrin in this non-canonical pathway. A human kidney tubular cell line in which ß1-integrin was knocked down (ß1-k/d) demonstrated enhanced type I collagen mRNA expression and promoter activity. A second shRNA to either αv-integrin or ß3-integrin, but not to another αv-binding partner, ß6-integrin, abrogated the enhanced COL1A2 promoter activity in ß1-k/d cells. Although αvß3-integrin surface expression levels were not different, αvß3-integrins colocalized with sites of focal adhesion significantly more in ß1-k/d cells, and activated αvß3-integrin was detected only in ß1-k/d cells. Further, the collagen response was decreased by a function-blocking antibody or a peptide inhibitor of αvß3-integrin. In cells lacking αvß3-integrin, the responses were attenuated, whereas the response was enhanced in αvß3-overexpressing cells. Rac1 and ERK, previously defined mediators for this non-canonical pathway, showed increased activities in ß1-k/d cells. Finally, inhibition of αvß3-integrin decreased Rac1 activity and COL1A2 promoter activity in ß1-k/d cells. Together, our results indicate that decreasing ß1 chain causes αvß3-integrin to become functionally dominant and promotes renal cell fibrogenesis via Rac1-mediated ERK activity.

Interdependence of HIF-1α and TGF-ß/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression.

Increasing evidence suggests that chronic kidney disease may develop following acute kidney injury and that this may be due, in part, to hypoxia-related phenomena. Hypoxia-inducible factor (HIF) is stabilized in hypoxic conditions and regulates multiple signaling pathways that could contribute to renal fibrosis. As transforming growth factor (TGF)-ß is known to mediate renal fibrosis, we proposed a profibrotic role for cross talk between the TGF-ß1 and HIF-1α signaling pathways in kidney cells. Hypoxic incubation increased HIF-1α protein expression in cultured human renal tubular epithelial cells and mouse embryonic fibroblasts. TGF-ß1 treatment further increased HIF-1α expression in cells treated with hypoxia and also increased HIF-1α in normoxic conditions. TGF-ß1 did not increase HIF-1α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances normoxic HIF-1α translation. TGF-ß receptor (ALK5) kinase activity was required for increased HIF-1α expression in response to TGF-ß1, but not to hypoxia. A dominant negative Smad3 decreased the TGF-ß-stimulated reporter activity of a HIF-1α-sensitive hypoxia response element. Conversely, a dominant negative HIF-1α construct decreased Smad-binding element promoter activity in response to TGF-ß. Finally, blocking HIF-1α transcription with a biochemical inhibitor, a dominant negative construct, or gene-specific knockdown decreased basal and TGF-ß1-stimulated type I collagen expression, while HIF-1α overexpression increased both. Taken together, our data demonstrate cooperation in signaling between Smad3 and HIF-1α and suggest a new paradigm in which HIF-1α is necessary for normoxic, TGF-ß1-stimulated renal cell fibrogenesis.

Role of SARA (SMAD anchor for receptor activation) in maintenance of epithelial cell phenotype.

By inducing epithelial-to-mesenchymal transition (EMT), transforming growth factor-beta (TGF-beta) promotes cancer progression and fibrosis. Here we show that expression of the TGF-beta receptor-associated protein, SARA (Smad anchor for receptor activation), decreases within 72 h of exposure to TGF-beta and that this decline is both required and sufficient for the induction of several markers of EMT. It has been suggested recently that expression of the TGF-beta signaling mediators, Smad2 and Smad3, may have different functional effects, with Smad2 loss being more permissive for EMT progression. We find that the loss of SARA expression leads to a concomitant decrease in Smad2 expression and a disruption of Smad2-specific transcriptional activity, with no effect on Smad3 signaling or expression. Further, the effects of inducing the loss of Smad2 mimic those of the loss of SARA, enhancing expression of the EMT marker, smooth muscle alpha-actin. Smad2 mRNA levels are not affected by the loss of SARA. However, the ubiquitination of Smad2 is increased in SARA-deficient cells. We therefore examined the E3 ubiquitin ligase Smurf2 and found that although Smurf2 expression was unaltered in SARA-deficient cells, the interaction of Smad2 and Smurf2 was enhanced. These results describe a significant role for SARA in regulating cell phenotype and suggest that its effects are mediated through modification of the balance between Smad2 and Smad3 signaling. In part, this is achieved by enhancing the association of Smad2 with Smurf2, leading to Smad2 degradation.

A conceptual framework for the molecular pathogenesis of progressive kidney disease.

The data regarding the pathogenesis of progressive kidney disease implicate cytokine effects, physiological factors, and myriad examples of relatively nonspecific cellular dysfunction. The sheer volume of information being generated on this topic threatens to overwhelm our efforts to understand progression in chronic kidney disease or to derive rational strategies to treat it. Here, a conceptual framework is offered for organizing and considering these data. Disease is initiated by an injury that evokes a tissue-specific cellular response. Subsequent structural repair may be effective, or the new structure may be sufficiently changed that it requires an adaptive physiological response. If this adaptation is not successful, subsequent cycles of misdirected repair or maladaptation may lead to progressive nephron loss. To illustrate how this framework can be used to organize our approach to disease pathogenesis, the role of cytokines in proteinuria and progressive glomerular disease is discussed. Finally, this theoretical framework is reconsidered to examine its implications for the diagnosis and treatment of clinical conditions. Application of this schema could have significant relevance to both research inquiry and clinical practice.

Immediate impact of the COVID-19 pandemic on CTSA TL1 and KL2 training and career development.

Clinical and Translational Science Award (CTSA) TL1 trainees and KL2 scholars were surveyed to determine the immediate impact of the COVID-19 pandemic on training and career development. The most negative impact was lack of access to research facilities, clinics, and human subjects, plus for KL2 scholars lack of access to team members and need for homeschooling. TL1 trainees reported having more time to think and write. Common strategies to maintain research productivity involved time management, virtual connections with colleagues, and shifting to research activities not requiring laboratory/clinic settings. Strategies for mitigating the impact of the COVID-19 pandemic on training and career development are described.

Rac1 promotes TGF-beta-stimulated mesangial cell type I collagen expression through a PI3K/Akt-dependent mechanism.

Transforming growth factor (TGF)-beta is a central mediator in the progression of glomerulosclerosis, leading to accumulation of aberrant extracellular matrix proteins and inappropriate expression of smooth muscle alpha-actin in the kidney. Previously, we reported that disrupting the cytoskeleton diminished TGF-beta-stimulated type I collagen accumulation in human mesangial cells. As cytoskeletal signaling molecules, including the Rho-family GTPases, have been implicated in fibrogenesis, we sought to determine the respective roles of RhoA and Rac1 in HMC collagen I expression. TGF-beta1 activated both RhoA and Rac1 within 5 min of treatment, and this activation was dependent on the kinase activity of the type I TGF-beta receptor. TGF-beta1-stimulated induction of type I collagen mRNA expression and promoter activity was diminished by inhibiting Rac1 activity and was increased by a constitutively active Rac1 mutant, whereas inhibiting RhoA activity had no such effect. Rac1 activation required phosphatidylinositol-3-kinase (PI3K) activity. Furthermore, the PI3K antagonist, LY294002, reduced TGF-beta1-stimulated COL1A2 promoter activity and Rac1 activation. It also partially blocked active Rac1-stimulated collagen promoter activity, suggesting that PI3K activity contributes to both TGF-beta activation of Rac1 and signal propagation downstream of Rac1. Thus, while both Rac1 and RhoA are rapidly activated in response to TGF-beta1 in human mesangial cells, only Rac1 activation enhances events that contribute to mesangial cell collagen expression, through a positive feedback loop involving PI3K.

High ambient glucose augments angiotensin II-induced proinflammatory gene mRNA expression in human mesangial cells: effects of valsartan and simvastatin.

BACKGROUND: Hyperglycemia may potentiate the adverse renal effects of angiotensin II (AII). In the kidney, the major target of AII action is the glomerular mesangial cell, where its hemodynamic and proinflammatory action contributes to renal injury. AII action is mediated by several types of cell receptors. Among those, the AT1 receptor has been best studied using specific AII receptor blockers (ARBs). These agents have emerged as major new modalities in the prevention and amelioration of renal disease where the ARB renoprotective anti-inflammatory properties could be more important than previously appreciated. Like the ARBs, statins may also modulate inflammatory responses that are renoprotective and complement their cholesterol-lowering effects. AIM: The aim of this project was to (i) identify a repertoire of proinflammatory mesangial cell AII-inducible mRNAs; (ii) determine if the AII-induced proinflammatory mRNA responses depend on ambient glucose, and (iii) test the anti-inflammatory effectiveness of an ARB, valsartan, either alone or in combination with a statin, simvastatin. RESULTS/CONCLUSIONS: Using high-density microarrays and real-time PCR we identified several AII-inducible proinflammatory mesangial genes that exhibited augmented mRNA responses in high-glucose milieu. Valsartan blocked the AII-induced mRNA expression of proinflammatory genes (i.e. MCP-1, LIF and COX-2) maintained in normal and high glucose. These observations add to the mounting evidence that ARBs have anti-inflammatory effects in the kidney, a beneficial effect that may be more important in protecting renal function in diabetic patients. While simvastatin inhibited expression of some mRNAs encoding chemokines/cytokines, it enhanced expression of mRNA encoding COX-2, a key mediator of inflammation. Thus, the non-cholesterol effects of statins on inflammatory responses appear complex.