Adult human kidney organoids originate from CD24+ cells and represent an advanced model for adult polycystic kidney disease.

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+ epithelial subpopulation. Long-term-cultured CD24+ cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR-Cas9 gene-edited PKD1- and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling.

Identification of platelet-derived growth factor C as a mediator of both renal fibrosis and hypertension.

Platelet-derived growth factors (PDGF) have been implicated in kidney disease progression. We previously found that PDGF-C is upregulated at sites of renal fibrosis and that antagonism of PDGF-C reduces fibrosis in the unilateral ureteral obstruction model. We studied the role of PDGF-C in collagen 4A3-/- ("Alport") mice, a model of progressive renal fibrosis with greater relevance to human kidney disease. Alport mice were crossbred with PDGF-C-/- mice or administered a neutralizing PDGF-C antibody. Both PDGF-C deficiency and neutralization reduced serum creatinine and blood urea nitrogen levels and mitigated glomerular injury, renal fibrosis, and renal inflammation. Unexpectedly, systolic blood pressure was also reduced in both Alport and wild-type mice treated with a neutralizing PDGF-C antibody. Neutralization of PDGF-C reduced arterial wall thickness in the renal cortex of Alport mice. Aortic rings isolated from anti-PDGF-C-treated wildtype mice exhibited reduced tension and faster relaxation than those of untreated mice. In vitro, PDGF-C upregulated angiotensinogen in aortic tissue and in primary hepatocytes and induced nuclear factor κB (NFκB)/p65-binding to the angiotensinogen promoter in hepatocytes. Neutralization of PDGF-C suppressed transcript expression of angiotensinogen in Alport mice and angiotensin II receptor type 1 in Alport and wildtype mice. Finally, administration of neutralizing PDGF-C antibodies ameliorated angiotensin II-induced hypertension in healthy mice. Thus, in addition to its key role in mediating renal fibrosis, we identified PDGF-C as a mediator of hypertension via effects on renal vasculature and on the renin-angiotensin system. The contribution to both renal fibrosis and hypertension render PDGF-C an attractive target in progressive kidney disease.

Cold Shock Proteins Mediate GN with Mesangioproliferation.

DNA binding protein A (DbpA) is a member of the human cold shock domain-containing protein superfamily, with known functions in cell proliferation, differentiation, and stress responses. DbpA mediates tight junction-associated activities in tubular epithelial cells, but the function of DbpA in mesangial cells is unknown. Here, we found DbpA protein expression restricted to vascular smooth muscle cells in healthy human kidney tissue but profound induction of DbpA protein expression within the glomerular mesangial compartment in mesangioproliferative nephritis. In vitro, depletion or overexpression of DbpA using lentiviral constructs led to inhibition or promotion, respectively, of mesangial cell proliferation. Because platelet-derived growth factor B (PDGF-B) signaling has a pivotal role in mesangial cell proliferation, we examined the regulatory effect of PDGF-B on DbpA. In vitro studies of human and rat mesangial cells confirmed a stimulatory effect of PDGF-B on DbpA transcript numbers and protein levels. Additional in vivo investigations showed DbpA upregulation in experimental rat anti-Thy1.1 nephritis and murine mesangioproliferative nephritis models. To interfere with PDGF-B signaling, we injected nephritic rats with PDGF-B neutralizing aptamers or the MEK/ERK inhibitor U0126. Both interventions markedly decreased DbpA protein expression. Conversely, continuous PDGF-B infusion in healthy rats induced DbpA expression predominantly within the mesangial compartment. Taken together, these results indicate that DbpA is a novel target of PDGF-B signaling and a key mediator of mesangial cell proliferation.

Role of platelet-derived growth factor-CC in capillary rarefaction in renal fibrosis.

We have identified platelet-derived growth factor (PDGF)-CC as a potent profibrotic mediator in kidney fibrosis and pro-angiogenic mediator in glomeruli. Because renal fibrosis is associated with progressive capillary rarefaction, we asked whether PDGF-CC neutralization in fibrosis might have detrimental anti-angiogenic effects leading to aggravated peritubular capillary loss. We analyzed capillary rarefaction in mice with and without PDGF-CC neutralization (using genetically deficient mice and neutralizing antibodies), in three different models of renal interstitial fibrosis, unilateral ureteral obstruction, unilateral ischemia-reperfusion, Col4a3-deficient (Alport) mice, and healthy animals. Independent of the effect of PDGF-CC neutralization on renal fibrosis, we found no difference in capillary rarefaction between PDGF-CC-neutralized mice and mice with intact PDGF-CC. We also found no differences in microvascular leakage (determined by extravasation of Evans Blue Dye) and in renal relative blood volume quantified using in vivo microcomputed tomography. PDGF-CC neutralization had no effects on renal microvasculature in healthy animals. Capillary endothelium did not express PDGF receptor-α, suggesting that potential PDGF-CC effects would have to be indirect. PDGF-CC neutralization or deficiency was not associated with preservation or accelerated loss of peritubular capillaries, suggesting no significant pro-angiogenic effects of PDGF-CC during renal fibrosis. From a clinical perspective, the profibrotic effects of PDGF-CC outweigh the pro-angiogenic effects and, thus, do not limit a potential therapeutic use of PDGF-CC inhibition in renal fibrosis.

Urinary exosomes: a novel means to non-invasively assess changes in renal gene and protein expression.

BACKGROUND: In clinical practice, there is a lack of markers for the non-invasive diagnosis and follow-up of kidney disease. Exosomes are membrane vesicles, which are secreted from their cells of origin into surrounding body fluids and contain proteins and mRNA which are protected from digestive enzymes by a cell membrane. METHODS: Toxic podocyte damage was induced by puromycin aminonucleoside in rats (PAN). Urinary exosomes were isolated by ultracentrifugation at different time points during the disease. Exosomal mRNA was isolated, amplified, and the mRNA species were globally assessed by gene array analysis. Tissue-specific gene and protein expression was assessed by RT-qPCR analysis and immunohistochemistry. RESULTS: Gene array analysis of mRNA isolated from urinary exosomes revealed cystatin C mRNA as one of the most highly regulated genes. Its gene expression increased 7.5-fold by day 5 and remained high with a 1.9-fold increase until day 10. This was paralleled by a 2-fold increase in cystatin C mRNA expression in the renal cortex. Protein expression in the kidneys also dramatically increased with de novo expression of cystatin C in glomerular podocytes in parts of the proximal tubule and the renal medulla. Urinary excretion of cystatin C increased approximately 2-fold. CONCLUSION: In this proof-of-concept study, we could demonstrate that changes in urinary exosomal cystatin C mRNA expression are representative of changes in renal mRNA and protein expression. Because cells lining the urinary tract produce urinary exosomal cystatin C mRNA, it might be a more specific marker of renal damage than glomerular-filtered free cystatin C.

Platelet-derived growth factors (PDGFs) in glomerular and tubulointerstitial fibrosis.

Renal fibrosis is the hallmark of chronic kidney disease progression and is characterized by an exaggerated wound-healing process with the production of renal scar tissue. It comprises both the glomerular and the tubulointerstitial compartments. Among the factors that contribute to kidney fibrosis, the members of the platelet-derived growth factor (PDGF) family are among the best characterized ones. They appear to be the key factors in driving renal fibrosis, independent of the underlying kidney disease. The PDGF family consists of four isoforms (PDGF-A, -B, -C, and -D) and two receptor chains (PDGFR-α and -ß), which are constitutively or inducibly expressed in most renal cells. These components have an irreplaceable role in kidney development by recruitment of mesenchymal cells to the glomerular and tubulointerstitial compartments. They further regulate multiple pathophysiologic processes including cell proliferation, cell migration, expression and accumulation of extracellular matrix, production and secretion of pro- and anti-inflammatory mediators, vascular permeability, and hemodynamics. This review provides a brief update on the role of different PDGF isoforms in the development of glomerulosclerosis and tubulointerstitial fibrosis, newly identified endogeneous PDGF antagonists, and resulting potential therapies.

Cold shock Y-box protein-1 proteolysis autoregulates its transcriptional activities.

BACKGROUND: The Y-box protein-1 (YB-1) fulfills pleiotropic functions relating to gene transcription, mRNA processing, and translation. It remains elusive how YB-1 shuttling into the nuclear and cytoplasmic compartments is regulated and whether limited proteolysis by the 20S proteasome releases fragments with distinct function(s) and subcellular distribution(s). RESULTS: To address these questions, mapping of domains responsible for subcellular targeting was performed. Three nuclear localization signals (NLS) were identified. NLS-1 (aa 149-156) and NLS-2 (aa 185-194) correspond to residues with unknown function(s), whereas NLS-3 (aa 276-292) matches with a designated multimerization domain. Nuclear export signal(s) were not identified. Endoproteolytic processing by the 20S proteasome before glycine 220 releases a carboxy-terminal fragment (CTF), which localized to the nucleus, indicating that NLS-3 is operative. Genotoxic stress induced proteolytic cleavage and nuclear translocation of the CTF. Co-expression of the CTF and full-length YB-1 resulted in an abrogated transcriptional activation of the MMP-2 promoter, indicating an autoregulatory inhibitory loop, whereas it fulfilled similar trans-repressive effects on the collagen type I promoter. CONCLUSION: Compartmentalization of YB-1 protein derivatives is controlled by distinct NLS, one of which targets a proteolytic cleavage product to the nucleus. We propose a model for an autoregulatory negative feedback loop that halts unlimited transcriptional activation.

Albumin is recycled from the primary urine by tubular transcytosis.

Under physiologic conditions, significant amounts of plasma protein pass the renal filter and are reabsorbed by proximal tubular cells, but it is not clear whether the endocytosed protein, particularly albumin, is degraded in lysosomes or returned to the circulatory system intact. To resolve this question, a transgenic mouse with podocyte-specific expression of doxycycline-inducible tagged murine albumin was developed. To assess potential glomerular backfiltration, two types of albumin with different charges were expressed. On administration of doxycycline, podocytes expressed either of the two types of transgenic albumin, which were secreted into the primary filtrate and reabsorbed by proximal tubular cells, resulting in serum accumulation. Renal transplantation experiments confirmed that extrarenal transcription of transgenic albumin was unlikely to account for these results. Genetic deletion of the neonatal Fc receptor (FcRn), which rescues albumin and IgG from lysosomal degradation, abolished transcytosis of both types of transgenic albumin and IgG in proximal tubular cells. In summary, we provide evidence of a transcytosis within the kidney tubular system that protects albumin and IgG from lysosomal degradation, allowing these proteins to be recycled intact.

Biological responses to PDGF-AA versus PDGF-CC in renal fibroblasts.

BACKGROUND: Platelet-derived growth factors (PDGF)-AA and -CC mediate renal fibroblast proliferation and/or renal fibrosis. Whereas PDGF-CC binds to both the PDGF receptors (PDGFRs)-αα- and -αß, PDGF-AA binds more selectively to the αα-receptor, suggesting potential differences in the biological activities. METHODS: We compared signal transduction, gene expression as well as changes in the proteome induced by PDGF-AA and -CC in rat renal fibroblasts, which express both PDGFR subunits. The growth factor concentrations used were chosen based on their equipotency in inducing rat renal fibroblast proliferation. RESULTS: Both PDGF-AA and PDGF-CC induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Renal fibroblast proliferation induced by either PDGF-AA or -CC could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase (MAPK)-, Janus-kinase (JAK)/signal transducers and activators of transcription (STAT) and phosphatidyl-inositol-3-kinase (PI3K) pathway, pointing to the involvement of all the three pathways. However, quantitative differences between both the stimulations were minor. Additive or synergistic effects by stimulating simultaneously with PDGF-AA and -CC were not observed. Using a proteomic approach we found eleven differentially expressed proteins, which were quantitatively altered after treatment with either PDGF-AA or PDGF-CC. The regulation of calreticulin and inorganic pyrophosphatase 1 could be verified by western blotting. CONCLUSIONS: PDGF-AA and -CC exhibit almost identical biological effects on signal transduction and proteome in cultured renal fibroblasts, suggesting that the ligands exert their activity essentially through the commonly bound PDGFR-αα. Nonetheless, two differentially expressed proteins were identified which might be involved in the development of renal failure.

Platelet-derived growth factor (PDGF)-C neutralization reveals differential roles of PDGF receptors in liver and kidney fibrosis.

Platelet-derived growth factors (PDGF) are key mediators of organ fibrosis. We investigated whether PDGF-C(-/-) mice or mice treated with neutralizing PDGF-C antibodies are protected from bile duct ligation-induced liver fibrosis, and we compared the effects with those of PDGF-C deficiency or neutralization on kidney fibrosis induced by unilateral ureteral obstruction. Unexpectedly, and in contrast to kidney fibrosis, PDGF-C deficiency or antagonism did not protect from liver fibrosis or functional liver impairment. Furthermore, the hepatic infiltration of monocytes/macrophages/dendritic cells and chemokine mRNA expression (CC chemokine ligand [CCL]5, CCL2, and CC chemokine receptor 2 [CCR2]) remained unchanged. Transcript expression of PDGF ligands increased in both liver and kidney fibrosis and was not affected by neutralization of PDGF-C. In kidney fibrosis, PDGF-C deficiency or antagonism led to reduced expression and signaling of PDGF-receptor (R)-α- and PDGFR-ß-chains. In contrast, in liver fibrosis there was either no difference (PDGF-C(-/-) mice) or even an upregulation of PDGFR-ß and signaling (anti-PDGF-C group). Finally, in vitro studies in portal myofibroblasts pointed to a predominant role of PDGF-B and PDGF-D signaling in liver fibrosis. In conclusion, our study revealed significant differences between kidney and liver fibrosis in that PDGF-C mediates kidney fibrosis, whereas antagonism of PDGF-C in liver fibrosis appears to be counteracted by significant upregulation and increased PDGFR-ß signaling. PDGF-C antagonism, therefore, may not be effective to treat liver fibrosis.

Growth arrest-specific protein 1 is a novel endogenous inhibitor of glomerular cell activation and proliferation.

Growth arrest-specific protein-1 (GAS1) is a GPI-anchored protein which is highly expressed in embryonic mouse fibroblasts and inhibits their proliferation. Glomerular mesangial cells release soluble GAS1 protein into the supernatant in vitro. Growth arrest led to GAS1 overexpression and increased release. Secretion involved disintegrin and metalloproteinase 10 and 17 as signified by inhibition experiments. Recombinant soluble GAS1 protein inhibited the proliferation of mesangial cells. Conversely, the induction of mesangial cell proliferation by PDGF-BB or -DD led to downregulation of GAS1 mRNA. Specific ligands of the PDGF α-receptor, PDGF-AA and -CC, had no effect. The GAS1 protein was localized in podocytes in kidneys from healthy rats. During the time course of mesangioproliferative glomerulonephritis in anti-Thy1.1-treated rats, glomerular GAS1 expression decreased prior to the onset of mesangial cell proliferation and increased at later stages during glomerular recovery. Finally, a plasmid expressing soluble GAS1 fused to an Fc fragment was systemically overexpressed in rats with mesangioproliferative glomerulonephritis. This ameliorated renal damage was indicated by decreased albuminuria and serum creatinine. Gas1/Fc-transfected rats also exhibited a reduction of the glomerular mesangial cell activation and proliferation. Thus, GAS1 is a novel endogenous inhibitor of glomerular mesangial cell proliferation and may be a novel therapeutic target in mesangioproliferative glomerular diseases.

Late angiotensin II receptor blockade in progressive rat mesangioproliferative glomerulonephritis: new insights into mechanisms.

Mesangioproliferative glomerulonephritis is the most common nephritis worldwide. We examined the effects of low- and high-dose telmisartan, an angiotensin II receptor blocker, in rats with progressive anti-Thy1.1 mesangioproliferative glomerulonephritis in a clinically relevant situation of established renal damage. Uninephrectomized nephritic rats were randomized on day 28 to remain untreated (control treatment; CT), or to receive low- (0.1 mg/kg/day, LT) or high-dose telmisartan (10 mg/kg/day, HT), hydrochlorothiazide + hydralazine (8 + 32 mg/kg/day, HCT + H), or atenolol (100 mg/kg/day, AT). CT and LT rats were hypertensive, whereas HT, HCT + H and AT treatment normalized blood pressures. On day 131, despite similar blood lowering effects, only HT, but not AT or HCT + H, prevented loss of renal function and reduced proteinuria compared to CT. Only HT potently ameliorated glomerulosclerosis, tubulointerstitial damage, cortical matrix deposition, podocyte damage and macrophage infiltration. HT reduced cortical expression of platelet derived growth factor receptor-α and -ß as well as transforming growth factor-ß1. LT exhibited minor but significant efficacy even in the absence of antihypertensive effects. Transcript array analyses revealed a four-fold down-regulation of renal cortical chemokine (C-C motif) receptor 6 (CCR6) mRNA by HT, which was confirmed at the protein level. Silencing of CCR6 did not alter podocyte function in vitro, thus indicating a predominant role in the tubulo-interstitium. In human kidney biopsies, CCR6 mRNA and mRNA of its ligand chemokine (C-C motif) ligand 20 was up-regulated in patients with progressive IgA nephropathy compared to stable disease. Thus, delayed treatment with high-dose telmisartan exerted a pronounced benefit in progressive mesangioproliferative glomerulonephritis, which extended beyond that of equivalent blood pressure lowering. We identified down-regulation of platelet-derived growth factor receptors and CCR6 as potential mediators of telmisartan-related renoprotection. CCR6 may also regulate the renal outcome in human mesangioprolfierative glomerulonephritis.

A novel, dual role of CCN3 in experimental glomerulonephritis: pro-angiogenic and antimesangioproliferative effects.

In contrast to factors that promote mesangial cell proliferation, little is known about their endogenous inhibitors. During experimental mesangioproliferative nephritis, expression of the glomerular CCN3 (nephroblastoma overexpressed gene [NOV]) gene is reduced before the proliferative phase and increased in glomeruli and serum when mesangial cell proliferation subsides. To further elucidate its role in mesangioproliferative glomerulonephritis, CCN3 systemically was overexpressed by muscle electroporation in healthy or nephritic rats. This increased CCN3 serum concentrations more than threefold for up to 56 days. At day 5 after disease induction, CCN3-transfected rats showed an increase in glomerular endothelial area and in mRNA levels of the pro-angiogenic factors vascular endothelial growth factor and PDGF-C. At day 7, CCN3 overexpression decreased mesangial cell proliferation, including expression of α-smooth muscle actin and matrix accumulation of fibronectin and type IV collagen. In progressive nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria, glomerulosclerosis, and reduced cortical collagen type I accumulation. In healthy rat kidneys, overexpression of CCN3 induced no morphologic changes but regulated glomerular gene transcripts (reduced transcription of PDGF-B, PDGF-D, PDGF-receptor-ß, and fibronectin, and increased PDGF-receptor-α and PDGF-C mRNA). These data identify a dual role for CCN3 in experimental glomerulonephritis with pro-angiogenic and antimesangioproliferative effects. Manipulation of CCN3 may represent a novel approach to help repair glomerular endothelial damage and mesangioproliferative changes.

The platelet-derived growth factor system in renal disease: an emerging role of endogenous inhibitors.

The platelet-derived growth factor (PDGF) family consists of four isoforms which are secreted as homodimers (PDGF-AA, PDGF-BB, PDGF-CC and PDGF-DD) or heterodimers (PDGF-AB), and two receptor chains (PDGFR-α and -ß). All members of the PDGF system are constitutively or inducibly expressed in renal cells and are involved in the regulation of cell proliferation and migration, the accumulation of extracellular matrix proteins and the secretion of pro- and anti-inflammatory mediators. Particular roles have been identified in mediating mesangioproliferative changes, renal interstitial fibrosis and glomerular angiogenesis. Different endogenous inhibitors of PDGF-induced biological responses exist which affect the activation/deactivation of PDGF isoforms, the activity of the PDGFRs, or which block downstream signaling pathways of the autophosphorylated PDGFRs. The novel endogenous inhibitor nephroblastoma overexpressed gene (NOV, CCN3) reduces PDGF-induced cell proliferation and is downregulated by PDGF isoforms itself. Among all identified inhibitors only few "true" PDGF antagonists have been identified. A better understanding of these inhibitors may aid in the design of novel therapeutic approaches to PDGF-mediated diseases.

Induction of progressive glomerulonephritis by podocyte-specific overexpression of platelet-derived growth factor-D.

Platelet-derived growth factor-D (PDGF-D), normally expressed in podocytes, mediates mesangial cell proliferation in vivo. To study this further, we created transgenic mice with podocyte-specific overexpression of PDGF-D. Hemizygous mice were grossly indistinguishable from wild-type littermates through 11 months of age; however, hemizygous mice older than 4 weeks commonly exhibited increased cell proliferation within the glomerular tuft. Many hemizygous mice also developed widespread segmental glomerulosclerosis and focal extracapillary proliferation with fibrin/fibrinogen deposition, extensive tubulointerstitial damage, proteinuria, and renal insufficiency. Electron microscopy found focal foot process effacement. Renal mRNA expression of podocin and nephrin, as well as the number of glomerular WT-1-positive cells, were significantly reduced in hemizygous compared to wild-type mice, indicating loss and/or dedifferentation of podocytes. PDGF-A, -B, and both PDGF receptor chain mRNAs, fibronectin, type IV collagen, RANTES, MCP-1, and CCR-2 mRNAs were all increased in the renal cortex of PDGF-D transgenic mice. Only 8.5% of newborn mice were homozygous overexpressors exhibiting a mortality rate of 37% at 4 weeks. Thus, podocyte-specific overexpression of PDGF-D caused mesangioproliferative disease, glomerulosclerosis, and crescentic glomerulonephritis. Hence, podocyte-specific growth factor overexpression can induce paracrine mesangial cell proliferation upstream of the filtration flow.

Effects and mechanisms of angiotensin II receptor blockade with telmisartan in a normotensive model of mesangioproliferative nephritis.

BACKGROUND: Renoprotective actions of angiotensin receptor blockers are not well established in normotensive, low-grade proteinuric glomerular diseases. We examined the effect of low-dose telmisartan (LT) and high-dose telmisartan (HT) versus conventional antihypertensive therapy in the rat anti-Thy1.1 model of glomerulonephritis. METHODS: Rats were randomized on Day 4 after disease induction to no treatment (CT, control), LT or HT or hydrochlorothiazide + hydralazine (HCT + H). RESULTS: All rats remained normotensive: HT and HCT + H reduced blood pressure by 15-20%. LT, HT and HCT + H reduced glomerular endothelial cell proliferation and glomerular and interstitial matrix deposition on Day 14. Only HT reduced podocyte damage and tubular cell dedifferentiation on Day 9 and mesangial cell activation on Day 14. By gene expression analysis arrays, we identified discs-large homolog 1 and angiopoietin-like 4 as potential mediators of the HT effects. In addition, we identified several pathways possibly related to the pleiotropic effects of HT, including growth factor signalling, mammalian target of rapamycin signalling, protein ubiquitination, the Wnt-beta catenin pathway and hypoxia signaling. CONCLUSIONS: In summary, treatment with HT, initiated after the induction of disease, ameliorates glomerular and tubulointerstitial damage. We provide the first comprehensive insight into the mechanisms underlying the renoprotective effect of high-dose angiotensin II receptor blockers (ARBs). Our study lays the basis for future investigations on novel pathways affected by ARBs in renal disease.

PDGF-C mediates glomerular capillary repair.

Glomerular endothelial cell injury is a key component of a variety of diseases. Factors involved in glomerular endothelial cell repair are promising therapeutic agents for such diseases. Platelet-derived growth factor (PDGF)-C has pro-angiogenic properties; however, nothing is known about such functions in the kidney. We therefore investigated the consequences of either PDGF-C infusion or inhibition in rats with mesangioproliferative glomerulonephritis, which is accompanied by widespread glomerular endothelial cell damage. We also assessed the role of PDGF-C in a mouse model of thrombotic microangiopathy as well as in cultured glomerular endothelial cells. PDGF-C infusion in nephritic rats significantly reduced mesangiolysis and microaneurysm formation, whereas glomerular endothelial cell area and proliferation increased. PDGF-C infusion specifically up-regulated glomerular fibroblast growth factor-2 expression. In contrast, antagonism of PDGF-C in glomerulonephritis specifically reduced glomerular endothelial cell area and proliferation and increased mesangiolysis. Similarly, PDGF-C antagonism in murine thrombotic microangiopathy aggravated the disease and reduced glomerular endothelial area. In conditionally immortalized glomerular endothelial cells, PDGF-C was mitogenic and induced a 27-fold up-regulation of fibroblast growth factor-2 mRNA. PDGF-C also exerted indirect pro-angiogenic effects, since it induced endothelial cell mitogens and pro-angiogenic factors in mesangial cells and macrophages. These results identify PDGF-C as a novel, potent pro-angiogenic factor in the kidney that can accelerate capillary healing in experimental glomerulonephritis and thrombotic microangiopathy.

Platelet-derived growth factor isoform expression in carbon tetrachloride-induced chronic liver injury.

Platelet-derived growth factor (PDGF) has an essential role in liver fibrogenesis, as PDGF-B and -D both act as potent mitogens on culture-activated hepatic stellate cells (HSCs). Induction of PDGF receptor type-beta (PDGFR beta) in HSC is well documented in single-dose carbon tetrachloride (CCl(4))-induced acute liver injury. Of the newly discovered isoforms PDGF-C and -D, only PDGF-D shows significant upregulation in bile duct ligation (BDL) models. We have now investigated the expression of PDGF isoforms and receptors in chronic liver injury in vivo after long-term CCl(4) treatment and demonstrated that isolated hepatocytes have the requisite PDGF signaling pathways, both in the naive state and when isolated from CCl(4)-treated rats. In vivo, PDGF gene expression showed upregulation of all PDGF isoforms and receptors, with values peaking at 4 weeks and decreasing to near basal levels by 8 and 12 weeks. Interestingly, PDGF-C increased significantly when compared to BDL-models. PDGF-A, PDGF-C and PDGF receptor type-alpha (PDGFR alpha) correlated closely with inflammation and steatosis. Immunohistochemistry revealed expression of PDGF-B, -C and -D in areas corresponding to centrilobular necrosis, inflammation and fibrosis, whereas PDGF-A localized in regenerative hepatocytes. PDGFR beta was identified along the fibrotic septa, whereas PDGFR alpha showed positive staining in fibrotic septa and regenerative hepatocytes. Despite a significant decline of PDGF isoforms, hepatocyte regeneration peaked at 8 weeks. A marked difference in the degree of fibrosis was observed amongst the individual animals. In summary, PDGF expression in liver damage primarily parallels mesenchymal cell proliferation and extracellular matrix production, rather than hepatocyte regeneration. We conclude that PDGF levels in chronic liver injury peak at 4 weeks after onset of injury, and that the outcome of chronic toxic liver injury strongly depends on the individual capacity for tissue regeneration in the weeks following the peak of PDGF expression.

Pro-fibrogenic potential of PDGF-D in liver fibrosis.

BACKGROUND/AIMS: We analyzed the expression of platelet-derived growth factor D (PDGF-D) in an experimental bile duct-ligated (BDL) rat model and assessed its biological function in cultured hepatic stellate cells (HSC) and myofibroblasts (MFB). METHODS: The mRNA for PDGF-A, -B, -C, -D and for PDGF receptor-alpha and -beta chains (PDGFRalpha and PDGFRbeta) in normal and fibrotic rat livers was assessed quantitatively. Protein levels of PDGF-D were quantified by immunoblotting and immunohistochemistry. RESULTS: The relative mRNA expression of all PDGF isoforms and receptors upregulated upon BDL and PDGF-A, -B and -D expression was significantly higher than that of PDGF-C. PDGF-D and PDGFRbeta protein also increased markedly. Immunostaining revealed that PDGF-D is localized along the fibrotic septa of the periportal- and perisinusoidal areas. Besides PDGF-B, PDGF-D is the second most potent PDGF isoform in PDGFRbeta signaling within HSC/MFB, evidenced by PDGFRbeta autophosphorylation and activation of the downstream signaling molecules ERK1/2-, JNK-, p38 MAPK, and PKB/Akt while PDGF-C effects were minimal. PDGF-D exerted mitogenic and fibrogenic effects in both cultured HSC and MFB comparable to PDGF-B but PDGF-A and -C showed only marginal fibrogenic effects. CONCLUSIONS: PDGF-D possesses potential pathogenetic properties for HSC activation and matrix remodeling in liver fibrosis.

Complement C5 mediates experimental tubulointerstitial fibrosis.

Renal fibrosis is the final common pathway of most progressive renal diseases. C5 was recently identified as a risk factor for liver fibrosis. This study investigated the role of C5 in the development of renal tubulointerstitial fibrosis by (1) induction of renal fibrosis in wild-type and C5(-/-) mice by unilateral ureteral ligation (UUO) and (2) investigation of the effects of a C5a receptor antagonist (C5aRA) in UUO. In C5(-/-) mice, when compared with wild-type controls, markers of renal fibrosis (Sirius Red, type I collagen, fibronectin, alpha-smooth muscle actin, vimentin, and infiltrating macrophages) were significantly reduced on day 5 of UUO. On day 10, fibronectin mRNA and protein expression were still reduced in the C5(-/-) mice. Cortical mRNA of all PDGF isoforms and of TGF-beta(1) (i.e., central mediators of renal disease) were significantly reduced in C5(-/-) mice when compared with controls. Renal tubular cell expression of the C5aR was sparse in normal cortex but markedly upregulated after UUO. Treatment of wild-type UUO mice with C5aRA also led to a significant reduction of cortical Sirius Red staining, fibronectin protein expression, and PDGF-B mRNA expression on day 5. Neither genetic C5 deficiency nor C5aRA treatment caused any histologic changes in the nonobstructed kidneys. In cultured murine cortical tubular cells, C5a stimulated production of TGF-beta(1), and this was inhibited by C5aRA. Using a combined genetic and pharmacologic approach, C5, in particular C5a, is identified as a novel profibrotic factor in renal disease and as a potential new therapeutic target.