Lucas L. Falke

Diverse origins of the myofibroblast—implications for kidney fibrosis

Fibrosis is the common end point of chronic kidney disease. The persistent production of inflammatory cytokines and growth factors leads to an ongoing process of extracellular matrix production that eventually disrupts the normal functioning of the organ. During fibrosis, the myofibroblast is commonly regarded as the predominant effector cell. Accumulating evidence has demonstrated a diverse origin of myofibroblasts in kidney fibrosis. Proposed major contributors of myofibroblasts include bone marrow-derived fibroblasts, tubular epithelial cells, endothelial cells, pericytes and interstitial fibroblasts; the published data, however, have not yet clearly defined the relative contribution of these different cellular sources. Myofibroblasts have been reported to originate from various sources, irrespective of the nature of the initial damage responsible for the induction of kidney fibrosis. Here, we review the possible relevance of the diversity of myofibroblast progenitors in kidney fibrosis and the implications for the development of novel therapeutic approaches. Specifically, we discuss the current status of preclinical and clinical antifibrotic therapy and describe targeting strategies that might help support resident and circulating cells to maintain or regain their original functional differentiation state. Such strategies might help these cells resist their transition to a myofibroblast phenotype to prevent, or even reverse, the fibrotic state.

Targeting CTGF, EGF and PDGF pathways to prevent progression of kidney disease

Chronic kidney disease (CKD) is a major health and economic burden with a rising incidence. During progression of CKD, the sustained release of proinflammatory and profibrotic cytokines and growth factors leads to an excessive accumulation of extracellular matrix. Transforming growth factor β (TGF-β) and angiotensin II are considered to be the two main driving forces in fibrotic development. Blockade of the renin–angiotensin–aldosterone system has become the mainstay therapy for preservation of kidney function, but this treatment is not sufficient to prevent progression of fibrosis and CKD. Several factors that induce fibrosis have been identified, not only by TGF-β-dependent mechanisms, but also by TGF-β-independent mechanisms. Among these factors are the (partially) TGF-β-independent profibrotic pathways involving connective tissue growth factor, epidermal growth factor and platelet-derived growth factor and their receptors. In this Review, we discuss the specific roles of these pathways, their interactions and preclinical evidence supporting their qualification as additional targets for novel antifibrotic therapies.

Nephronophthisis-Associated CEP164 Regulates Cell Cycle Progression, Apoptosis and Epithelial-to-Mesenchymal Transition

We recently reported that centrosomal protein 164 (CEP164) regulates both cilia and the DNA damage response in the autosomal recessive polycystic kidney disease nephronophthisis. Here we examine the functional role of CEP164 in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of CEP164 revealed an overall quicker cell cycle than control cells, although early S-phase was significantly longer. Follow-up FACS experiments with renal IMCD3 cells confirm that Cep164 siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type CEP164, but not disease-associated mutants. siRNA of CEP164 revealed a proliferation defect over time, as measured by CyQuant assays. The discrepancy between accelerated cell cycle and inhibited overall proliferation could be explained by induction of apoptosis and epithelial-to-mesenchymal transition. Reduction of CEP164 levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of Cep164 or overexpression of dominant negative mutant allele CEP164 Q525X induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with cep164 morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response in vivo. This study reveals a novel role for CEP164 in the pathogenesis of nephronophthisis, in which mutations cause ciliary defects coupled with DNA damage induced replicative stress, cell death, and epithelial-to-mesenchymal transition, and suggests that these events drive the characteristic fibrosis observed in nephronophthisis kidneys.

Loss of tumour suppressor PTEN expression in renal injury initiates SMAD3- and p53-dependent fibrotic responses

Deregulation of the tumour suppressor PTEN occurs in lung and skin fibrosis and diabetic and ischaemic renal injury. However, the potential role of PTEN and associated mechanisms in the progression of kidney fibrosis is unknown. Tubular and interstitial PTEN expression was dramatically decreased in several models of renal injury, including aristolochic acid nephropathy (AAN), streptozotocin (STZ)‐mediated injury and ureteral unilateral obstruction (UUO), correlating with Akt, p53 and SMAD3 activation and fibrosis. Stable silencing of PTEN in HK‐2 human tubular epithelial cells induced dedifferentiation and CTGF, PAI‐1, vimentin, α‐SMA and fibronectin expression, compared to HK‐2 cells expressing control shRNA. Furthermore, PTEN knockdown stimulated Akt, SMAD3 and p53Ser15 phosphorylation, with an accompanying decrease in population density and an increase in epithelial G1 cell cycle arrest. SMAD3 or p53 gene silencing or pharmacological blockade partially suppressed fibrotic gene expression and relieved growth inhibition orchestrated by deficiency or inhibition of PTEN. Similarly, shRNA suppression of PAI‐1 rescued the PTEN loss‐associated epithelial proliferative arrest. Moreover, TGFβ1‐initiated fibrotic gene expression is further enhanced by PTEN depletion. Combined TGFβ1 treatment and PTEN silencing potentiated epithelial cell death via p53‐dependent pathways. Thus, PTEN loss initiates tubular dysfunction via SMAD3‐ and p53‐mediated fibrotic gene induction, with accompanying PAI‐1‐dependent proliferative arrest, and cooperates with TGFβ1 to induce the expression of profibrotic genes and tubular apoptosis. Copyright

A perspective on anti-CCN2 therapy for chronic kidney disease

Kidney fibrosis is the common end point of chronic kidney disease independent of aetiology. Currently, no effective therapy exists to reduce kidney fibrosis. CCN2 appears to be an interesting candidate for anti-fibrotic drug targeting, because it holds a central position in the development of kidney fibrosis and interacts with a variety of factors that are involved in the fibrotic response, including transforming growth factor (TGF) β and Bone morphogenetic proteins. Although CCN2 modifies many pathways, it does not appear to have a membrane receptor of its own. Numerous experimental and clinical studies lowering CCN2 bioavailability have shown promising results with minimal adverse side effects. This review aims to provide an overview of the current state of CCN2 research with a focus on anti-fibrotic therapy.

Hemizygous deletion of CTGF/CCN2 does not suffice to prevent fibrosis of the severely injured kidney.

BACKGROUND Connective Tissue Growth Factor (CTGF/CCN2) is an important mediator of kidney fibrosis. Previous observations indicated that attenuation of CCN2 expression sufficed to alleviate early kidney damage. However, little is known about the role of CCN2 in fibrosis of severely damaged and more chronically injured kidneys. Therefore, we examined the effects of CCN2 haploinsufficiency on the progression of renal scarring in long-term STZ-induced diabetic nephropathy, in a more advanced stage of obstructive nephropathy following unilateral ureteric obstruction (UUO), and in severe aristolochic acid (AA)-induced tubulotoxic nephritis. METHODS Wild-type (WT, CCN2(+/+)) and hemizygous CCN2(+/-) C57Bl/6 mice were studied. In the diabetes experiment, streptozotocin-injected and control mice were followed for 6 months, with regular blood pressure, glycaemia and albuminuria recordings. In the UUO experiment, the left ureter was obstructed for 14 days with the contralateral kidney serving as control. For the AA experiment, mice were followed for 25 days after 5 intraperitoneal injections with AA and compared to control mice injected with buffer alone. Organs were harvested for histology, mRNA and protein measurements. Collagen content was determined by HPLC and expressed as hydroxyproline/proline ratio. RESULTS CCN2 expression was significantly increased in the damaged as compared to control kidneys. In all three models, CCN2 levels in the damaged kidneys of CCN2(+/-) mice averaged about 50% of those in damaged WT kidneys. After 6 months of diabetes, albuminuria was increased 2.5-fold in WT mice, compared to 1.5-fold in CCN2(+/-) mice, mesangial matrix was expanded 5-fold in WT and 4.4-fold in CCN2(+/-) mice and the glomerular basement membrane was thickened 1.3-fold in WT and 1.5-fold in CCN2(+/-) mice (all differences between WT and CCN2(+/-) mice are NS). Tubular damage and interstitial fibrosis scores were also not different between Wt and CCN2(+/-) mice in the diabetes (1.8 vs. 1.7), UUO (2.8 vs. 2.6), and AA (1.4 vs. 1.2) models, as was the case for macrophage influx and collagen content in these three models. CONCLUSION Unlike in mild and relatively early STZ-induced diabetic nephropathy, scarring of severely and chronically damaged kidneys is not attenuated by a 50% reduction of CCN2 to (near) normal levels. This suggests that CCN2 is either redundant in severe and chronic kidney disease, or that it is a limiting factor only at subnormal concentrations requiring further reduction by available or emerging therapies to prevent fibrosis of the severely injured kidney.

CTGF knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload

BACKGROUND One of the main contributors to maladaptive cardiac remodeling is fibrosis. Connective tissue growth factor (CTGF), a matricellular protein that is secreted into the cardiac extracellular matrix by both cardiomyocytes and fibroblasts, is often associated with development of fibrosis. However, recent studies have questioned the role of CTGF as a pro-fibrotic factor. Therefore, we aimed to investigate the effect of CTGF on cardiac fibrosis, and on functional, structural, and electrophysiological parameters in a mouse model of CTGF knockout (KO) and chronic pressure overload. METHODS AND RESULTS A new mouse model of global conditional CTGF KO induced by tamoxifen-driven deletion of CTGF, was subjected to 16weeks of chronic pressure overload via transverse aortic constriction (TAC, control was sham surgery). CTGF KO TAC mice presented with hypertrophic hearts, and echocardiography revealed a decrease in contractility on a similar level as control TAC mice. Ex vivo epicardial mapping showed a low incidence of pacing-induced ventricular arrhythmias (2/12 in control TAC vs. 0/10 in CTGF KO TAC, n.s.) and a tendency towards recovery of the longitudinal conduction velocity of CTGF KO TAC hearts. Picrosirius Red staining on these hearts unveiled increased fibrosis at a similar level as control TAC hearts. Furthermore, genes related to fibrogenesis were also similarly upregulated in both TAC groups. Histological analysis revealed an increase in fibronectin and vimentin protein expression, a significant reduction in connexin43 (Cx43) protein expression, and no difference in NaV1.5 expression of CTGF KO ventricles as compared with sham treated animals. CONCLUSION Conditional CTGF inhibition failed to prevent TAC-induced cardiac fibrosis and hypertrophy. Additionally, no large differences were found in other parameters between CTGF KO and control TAC mice. With no profound effect of CTGF on fibrosis formation, other factors or pathways are likely responsible for fibrosis development.

Local therapeutic efficacy with reduced systemic side effects by rapamycin-loaded subcapsular microspheres

Kidney injury triggers fibrosis, the final common pathway of chronic kidney disease (CKD). The increase of CKD prevalence worldwide urgently calls for new therapies. Available systemic treatment such as rapamycin are associated with serious side effects. To study the potential of local antifibrotic therapy, we administered rapamycin-loaded microspheres under the kidney capsule of ureter-obstructed rats and assessed the local antifibrotic effects and systemic side effects of rapamycin. After 7 days, microsphere depots were easily identifiable under the kidney capsule. Both systemic and local rapamycin treatment reduced intrarenal mTOR activity, myofibroblast accumulation, expression of fibrotic genes, and T-lymphocyte infiltration. Upon local treatment, inhibition of mTOR activity and reduction of myofibroblast accumulation were limited to the immediate vicinity of the subcapsular pocket, while reduction of T-cell infiltration was widespread. In contrast to systemically administered rapamycin, local treatment did not induce off target effects such as weight loss. Thus subcapsular delivery of rapamycin-loaded microspheres successfully inhibited local fibrotic response in UUO with less systemic effects. Therapeutic effect of released rapamycin was most prominent in close vicinity to the implanted microspheres.

Loss of expression of protein phosphatase magnesium-dependent 1A during kidney injury promotes fibrotic maladaptive repair.

Protein phosphatase magnesium‐dependent‐1A (PPM1A) dephosphorylates SMAD2/3, which suppresses TGF‐β signaling in keratinocytes and during Xenopus development; however, potential involvement of PPM1A in chronic kidney disease is unknown. PPM1A expression was dramatically decreased in the tubulointerstitium in obstructive and aristolochic acid nephropathy, which correlates with progression of fibrotic disease. Stable silencing of PPM1A in human kidney‐2 human renal epithelial cells increased SMAD3 phosphorylation, stimulated expression of fibrotic genes, induced dedifferentiation, and orchestrated epithelial cell‐cycle arrest via SMAD3‐mediated connective tissue growth factor and plasminogen activator inhibitor‐1 up‐regulation. PPM1A stable suppression in normal rat kidney‐49 renal fibroblasts, in contrast, promoted a SMAD3dependent connective tissue growth factor and plasminogen activator inhibitor‐1–induced proliferative response. Paracrine factors secreted by PPM1A‐depleted epithelial cells augmented fibroblast proliferation (>50%) compared with controls. PPM1A suppression in renal cells further enhanced TGF‐b1–induced SMAD3 phosphorylation and fibrotic gene expression, whereas PPM1A overexpression inhibited both responses. Moreover, phosphate tensin homolog on chromosome 10 depletion in human kidney‐2 cells resulted in loss of expression and decreased nuclear levels of PPM1A, which enhanced SMAD3‐mediated fibrotic gene induction and growth arrest that were reversed by ectopic PPM1A expression. Thus, phosphate tensin homolog on chromosome 10 is an upstream regulator of renal PPM1A deregulation. These findings establish PPM1A as a novel repressor of the SMAD3 pathway in renal fibrosis and as a new therapeutic target in patients with chronic kidney disease.—Samarakoon, R., Rehfuss, A., Khakoo, N.S., Falke, L. L., Dobberfuhl, A.D., Helo, S., Overstreet, J.M., Goldschmeding, R., Higgins, P. J. Loss of expression of protein phosphatase magnesium‐dependent 1A during kidney injury promotes fibrotic maladaptive repair. FASEBJ. 30, 3308–3320 (2016). www.fasebj.org

Cellular senescence in the aging and diseased kidney

The program of cellular senescence is involved in both the G1 and G2 phase of the cell cycle, limiting G1/S and G2/M progression respectively, and resulting in prolonged cell cycle arrest. Cellular senescence is involved in normal wound healing. However, multiple organs display increased senescent cell numbers both during natural aging and after injury, suggesting that senescent cells can have beneficial as well as detrimental effects in organismal aging and disease. Also in the kidney, senescent cells accumulate in various compartments with advancing age and renal disease. In experimental studies, forced apoptosis induction through the clearance of senescent cells leads to better preservation of kidney function during aging. Recent groundbreaking studies demonstrate that senescent cell depletion through INK-ATTAC transgene-mediated or cell-penetrating FOXO4-DRI peptide induced forced apoptosis, reduced age-associated damage and dysfunction in multiple organs, in particular the kidney, and increased performance and lifespan. Senescence is also involved in oncology and therapeutic depletion of senescent cells by senolytic drugs has been studied in experimental and human cancers. Although studies with senolytic drugs in models of kidney injury are lacking, their dose limiting side effects on other organs suggest that targeted delivery might be needed for successful application of senolytic drugs for treatment of kidney disease. In this review, we discuss (i) current understanding of the mechanisms and associated pathways of senescence, (ii) evidence of senescence occurrence and causality with organ injury, and (iii) therapeutic strategies for senescence depletion (senotherapy) including targeting, all in the context of renal aging and disease.