Chronic kidney disease (CKD) is a worldwide health problem affecting 9.1% of the world’s population. The treatments to prevent the progression of CKD remain limited, however. Resident fibroblasts in the kidneys play crucial roles in the pathological conditions commonly recognized in CKD, such as renal fibrosis, renal anemia, and peritubular capillary loss. Fibroblasts in the kidney provide structural backbone by producing extracellular matrix proteins and produce erythropoietin for normal hematopoiesis under physiological conditions. In the diseased condition, however, fibroblasts differentiate into myofibroblasts that produce excessive extracellular matrix proteins at the cost of the inherent erythropoietin-producing abilities, resulting in renal fibrosis and renal anemia. Pericytes, which are mesenchymal cells that enwrap peritubular capillaries and highly overlap with resident fibroblasts, detach from peritubular capillary walls in response to kidney injury, resulting in peritubular capillary loss and tissue hypoxia. Several reports have demonstrated the beneficial roles of fibroblasts in the regeneration of renal tubules Renal fibroblasts also have the potential to differentiate into a proinflammatory state, producing various cytokines and chemokines and prolonging inflammation by forming tertiary lymphoid tissues, functional lymphoid aggregates, in some pathological conditions. In this article, we describe the heterogenous functions of renal fibroblasts under healthy and diseased conditions.
- chronic kidney disease
- renal anemia
- tertiary lymphoid tissue
Chronic kidney disease (CKD) is a worldwide public health problem. In 2017, the prevalence of CKD was estimated to be 9.1% in the world’s population, and has increased by 29.3% from 1990 to 2017 . The prevalence of CKD in elderly individuals over 65 years old is especially high and is predicted to increase further as a result of the increasingly aged society . CKD is a risk factor for end-stage renal disease (ESRD) and is also recognized as an independent risk factor for cardiovascular diseases and their associated mortality . Patients with ESRD need renal replacement therapies such as dialysis and renal transplantation to survive. The cost of these therapies is enormous and the financial burden is a critical problem for patients and society . Nevertheless, treatment to prevent the progression of CKD and the occurrence of CKD-associated complications remain limited.
Fibroblasts are distributed in various organs throughout the body and contribute to both homeostasis and disease. In the kidney, resident fibroblasts play crucial roles in both health and disease, and their phenotypes are heterogenous and plastic . Under physiological conditions, renal fibroblasts provide structural support for the entire kidney architecture and produce erythropoietin (EPO). In contrast, in diseased kidneys, fibroblasts lose these physiological functions and transdifferentiate into myofibroblasts. These phenotypic changes result in fibrosis, renal anemia, and peritubular capillary loss, all of which are common pathological conditions of CKD, irrespective of the etiology . Renal fibroblasts also act as inflammatory cells and produce proinflammatory cytokines and chemokines under some pathological conditions [5, 7]. In aged injured kidneys, fibroblasts play a crucial role in prolonging inflammation by inducing tertiary lymphoid tissue (TLT) formation . These features highlight the importance of understanding the behavior of fibroblasts in the kidney in order to identify efficient therapeutic strategies to prevent CKD progression. In this article, we describe the current understanding of the heterogeneous functions of fibroblasts in healthy and diseased kidneys.
2. Fibroblasts in the kidney
2.1 Characteristics and functions of resident fibroblasts in kidneys
Renal resident fibroblasts are spindle-shaped cells that exist in the interstitial space, which is defined as the area between nephrons. Nephrons are functional units of the kidney and are composed of glomerular and tubular cells. Fibroblasts provide the structural backbone of the kidney by producing extracellular matrix (ECM) proteins and interact with surrounding cells to maintain the homeostatic state in healthy kidneys. Identification of resident fibroblasts is performed based on their location, shape, and positive expressions of several fibroblast markers such as CD73 and PDGFRβ . As these markers are neither homogeneously positive nor specific for resident fibroblasts, confirming the negative expression of other cell-lineage markers such as CD45, a hematopoietic cell marker, is also necessary to identify renal resident fibroblasts.
In addition to the role of structural cells, fibroblasts have unique organ-specific functions. In the kidney, small subset of the renal resident fibroblasts residing in the corticomedullary area produces Epo, a hormone essential for erythropoiesis in response to hypoxia . Although there are few Epo-producing cells and they exist only in the deep cortex in the physiological state, under severe hypoxic conditions such as severe anemia, the number of Epo-producing cells increases and they can be detected in the cortical area . The increase in the number of Epo-producing cells under hypoxic conditions is likely due to the increase in the number of the cells that have acquired Epo-producing ability but not to cellular proliferation, because Epo production is activated in anemic mice under administration of a cell cycle inhibitor or γ-ray irradiation . Interestingly, while other growth factors for hematopoietic cells (such as granulocyte colony-stimulating factor) are produced in bone marrow, where hematopoietic cells are generated and the growth factors are required, EPO is produced from the kidney. One possible explanation for this is that kidneys are physiologically hypoxic compared with other organs, which allows them to be more sensitive to small changes in oxygen delivery than other organs and is advantageous to the production of EPO in response to hypoxia . Another explanation is that kidneys function as a “critmeter,” with the ability to set hematocrit within the normal range by regulating plasma volume and the red blood cell mass in a common site .
2.2 Origin and heterogeneity of fibroblasts in the kidney
In 1974, Le Douarin et al. reported that, in transplantation experiments of quail neural tubes to chicks, quail neural crest–derived cells were identified in the renal interstitial space . Consistently, we found that fibroblasts in neonate kidneys express p75 neurotrophin receptor (p75NTR), a neural crest marker . Moreover, Epo-producing cells express neuronal markers such as microtubule-associated protein 2 and neurofilament light polypeptide . Based on these previous findings, we conducted a lineage tracing study using
2.3 Pericytes in kidneys
Pericytes are mesenchyme-derived cells that enwrap capillaries with their processes embedded in the vascular basement membrane. Resident fibroblasts and pericytes share several characteristics, including their interstitial location and cell surface markers such as CD73 and PDGFRβ, and, as such, these two types of cells are often confused. Pericytes support the capillary structure and regulate vascular tone with their contraction force . Moreover, they interact with endothelial cells to maintain capillary homeostasis . Humphreys et al. reported that the origins of pericytes in the kidneys were FoxD1-expressing cells in an experiment using
3. Renal fibrosis as a hallmark of CKD
Renal fibrosis is a common pathological condition of CKD, irrespective of the etiology. It is defined as excessive accumulation of ECM such as collagen and fibronectin in the interstitial space and is recognized as a predictive indicator of renal prognosis . Previous studies have shown that dysfunction of the renal fibroblasts can induce several pathological conditions associated with CKD, such as renal fibrosis, renal anemia, and peritubular capillary loss. Against this background, renal fibroblasts have been focused on as hopeful therapeutic targets for CKD and its complications.
3.1 Myofibroblasts in kidneys and their origin
Myofibroblasts are recognized as the main contributor to fibrosis in multiple organs. They are characterized by dense endoplasmic reticulum and contractile microfilament bundles . Their most prominent feature is the expression of α-smooth muscle actin (α-SMA) that forms myofilament bundles and promotes their high contractility . Although myofibroblasts are almost undetectable in healthy kidneys, they expand dramatically in diseased kidneys and drive fibrosis by producing a large amount of ECM proteins and through their own proliferation. The origin of myofibroblasts has been discussed for decades, and several genetic lineage tracing studies recently revealed that resident fibroblasts and pericytes are the main sources for myofibroblasts . We reported that
Notably, although genetic lineage tracing is not feasible in humans, a recent study utilizing scRNA-seq of human kidney samples supports the notion that these findings in mice appear to be conserved in humans. Kuppe et al. conducted scRNA-seq on human kidneys in patients with CKD and demonstrated that Notch3+ pericytes and Meg3+ fibroblasts were the main sources for highly ECM-producing myofibroblasts using pseudo-time trajectory analysis and diffusion map analysis . These studies support the idea that most renal myofibroblasts derive from renal resident fibroblasts or pericytes.
3.2 Progenitor of myofibroblasts; Gli1+ fibroblasts in the perivascular niche
Mesenchymal stem cells (MSCs) are defined as cells with self-renewal and clonogenic capacity. Gli1+ fibroblasts are MSC-like cells that reside in both the pericyte niche and the adventitia of larger vessels across multiple organs, including the kidney, and exhibit trilineage differentiation potential
3.3 The roles of proximal tubule injury in CKD progression
Acute kidney injury (AKI) is a highly prevalent disorder and is one of the risk factors for the progression of CKD . The underlying molecular mechanisms for CKD transition after AKI have been investigated for decades. The proximal tubules are the most vulnerable segment in the nephron, and are assumed to trigger the AKI to CKD progression. To investigate whether injured proximal tubules can trigger renal fibrosis, we selectively damaged proximal tubules by DT administration in
4. Two common CKD complications, renal anemia and peritubular capillary loss, are also caused by dysfunction of renal fibroblasts/pericytes
Renal anemia is a common complication that affects the majority of patients with CKD . The cause of renal anemia is the relative deficiency of EPO. Several recent studies have shown that dysfunction of renal fibroblasts contribute to this complication. EPO production is stimulated by hypoxia and regulated by hypoxia-inducible factors (HIFs). In normoxic conditions, HIFs are hydroxylated by HIF-prolyl hydroxylase domain–containing proteins (PHDs), and hydroxylated HIFs are degraded by the ubiquitin-proteasome system [36, 37]. In hypoxic conditions, the hydroxylation and degradation of HIFs is inhibited, resulting in the transcriptional activation of HIF-inducible genes, including
Although the administration of erythropoiesis-stimulating agents (ESAs) is a currently well-established and effective clinical treatment, it might be associated with several adverse effects, such as hypertension and thrombotic complications . To avoid safety concerns associated with ESAs, PHD inhibitors, which upregulate EPO production via the stabilization of HIFs, have been developed and used for the treatment of renal anemia [36, 42, 43, 44, 45].
Another common pathological feature of CKD is the loss of peritubular capillaries . Renal pericytes enwrap peritubular capillaries and support them structurally. In response to injury, pericytes detach from capillaries and their processes, which form networks surrounding the capillaries, start to direct from their associated capillaries to the adjacent tubules, concomitant with transdifferentiation into myofibroblasts . This pathological change makes peritubular capillaries unstable and causes capillary rarefaction and loss . Reduced peritubular capillary blood supply can cause chronic hypoxia in renal parenchymal cells such as tubules and stromal cells. Hypoxia aggravates renal fibrosis by stimulating fibroblasts and altering their gene expressions associated with ECM metabolism . For example, hypoxia upregulated collagen type 1 and the tissue inhibitor of metalloproteinase-1 expression and also downregulated matrix metalloproteinase-1
5. Beneficial function of myofibroblasts in kidneys
Contrary to the long-held assumption that fibrosis is detrimental to the host, recent evidence suggests that fibrosis also has host-protective roles in some cases. To investigate the role of fibroblasts during the early phase of kidney injury, we utilized
6. Renal fibroblasts associated with inflammation
Although myofibroblasts are established as the primary effector cells driving fibrosis, several recent studies have demonstrated that resident fibroblasts in the kidney also have a proinflammatory phenotype. Souma et al. reported that Epo-producing fibroblast–derived myofibroblasts upregulated the expression of the target genes of NFκB such as
It is of note that an anti-inflammatory role of fibroblasts and pericytes was also reported. Using
7. Age-dependent phenotype of fibroblasts: tertiary lymphoid tissue: associated fibroblasts
7.1 Tertiary lymphoid tissue formation in aged injured kidneys
An epidemiological study showed that elderly patients with AKI had an increased risk for CKD progression . The mechanism for the maladaptive repair after AKI in the elderly remains unknown. To identify the mechanism, we compared the renal response to injury between young and aged mice. As in humans, while young kidney repaired itself after injury, aged kidney exhibited sustained tubular injury and fibrosis . Unexpectedly, we found multiple TLTs in aged kidneys but not young kidneys in the chronic phase after kidney injury. TLT is an ectopic lymphoid tissue that develops at the site of chronic inflammation. TLTs are mainly composed of lymphocytes that are structurally and functionally supported by unique phenotypic fibroblasts inside TLTs (Figure 1). Unlike simple infiltration of the inflammatory cells, TLTs can promote lymphocyte proliferation and differentiation, resulting in the generation of antibody-secreting plasma cells, as recognized in secondary lymphoid organs . Importantly, although TLTs are identified in various disease conditions, such as autoimmune diseases, infections, and cancers, TLTs can play beneficial or pathological roles in a context-dependent manner . For example, in chronic inflammatory or autoimmune diseases, TLTs contribute to disease persistence and have detrimental effects on the host . In contrast, during infections, TLTs are assumed to play beneficial roles to eliminate pathogens by promoting immune responses . The role of TLTs in aged injured kidneys remains unclear, and will be discussed in the next section.
7.2 Characteristics and origin of fibroblasts inside tertiary lymphoid tissues
Fibroblasts inside TLTs exhibit unique characteristics that are distinct from those outside TLTs, such as the strong expression of p75NTR (Figure 1) . After kidney injury, resident fibroblasts acquire the ability to produce RAs by upregulating RALDH2, which is assumed to promote the transition of the adjacent fibroblasts into p75NTR+ TLT-associated fibroblasts. Some of the p75NTR+ TLT-associated fibroblasts acquire abilities to secrete homeostatic chemokines such as CCL19 and CXCL13, which are the driving force for recruiting lymphocytes and promoting TLT formation. Inside of more mature TLTs, CD21+/p75NTR− follicular dendritic cells (FDCs) appear as part of stromal cells (Figure 1). FDCs are stromal cells residing in the B cell follicles of secondary lymphoid organs; they drive germinal center reactions . These TLT-associated fibroblasts in the kidneys are
7.3 Clinical significance of tertiary lymphoid tissues in CKD and the elderly
Several studies have reported that TLTs are induced in various kidney diseases [62, 63, 64, 65]. Additionally, we reported that TLTs developed not only in murine kidneys but also in human kidneys in an age-dependent manner . In the analysis on kidneys from nephrectomy cases for renal cell carcinoma and autopsy, excluding pyelonephritis, glomerulonephritis, autoimmune kidney diseases, and hematological malignancies, TLTs were identified only in the elderly over 60 years old. The components of human TLTs are quite similar to those of murine TLTs. To evaluate TLTs objectively, we classified renal TLTs into three stages based on the immunostaining patterns as follows . TLTs not containing CD21+ FDCs or a germinal center response, dense Ki67+ proliferative B cell clusters, were defined as stage 1. TLTs containing CD21+ FDCs but no germinal center response were defined as stage 2. TLTs containing both CD21+ FDCs and a germinal center response were defined as stage 3. In this classification, the severity of the TLT stages and the area of TLTs were related with the severity of ischemic injury in murine renal IRI models. In humans, more and higher-stage TLTs were identified in the kidneys of patients with CKD than without CKD among elderly patients 60 years or older in the analysis using kidneys from nephrectomy cases due to renal cell carcinoma . These data demonstrated that the developmental stage of TLTs was associated with the severity of kidney injury, thereby indicating that TLTs have potential as a marker of severity of renal injury.
7.4 Potential of tertiary lymphoid tissues as therapeutic targets for CKD
Although TLTs are assumed to be associated with the severity of renal injury, it has been challenging to determine whether renal TLTs are pathogenic and if they directly affect renal function. We reported that, in unilateral renal IRI models of aged mice, the administration of GK1.5, anti-CD4 monoclonal antibody, diminished TLT formation and inflammatory marker expressions and improved renal fibrosis . This result suggests that renal TLTs could be pathogenic and the therapies targeting renal TLTs thus have the potential to improve renal function in patients with CKD. As this intervention is not specific to TLTs and affects systemic immune systems, however, a more specific therapy for TLTs is necessary to determine whether TLTs in aged injured kidneys are detrimental or not.
Resident fibroblasts in the kidney are essential components to maintain homeostasis under physiological conditions. In CKD, dysfunction of renal fibroblasts causes the main pathological conditions of renal fibrosis, renal anemia, and peritubular capillary loss. Importantly, renal fibroblasts are heterogeneous and have the potential to change their phenotypes depending on the local microenvironment (Figure 2) . Although myofibroblasts mainly contribute to renal fibrosis and deteriorate renal function by producing excessive ECM, they can also have host-protective roles in the early phase of kidney injury. Renal fibroblasts can differentiate into proinflammatory fibroblasts that secrete inflammatory cytokines and chemokines, which can promote TLT formation under several diseased conditions. Fibroblasts or pericytes also have an anti-inflammatory function via CD73 expression. A better understanding of the heterogeneity and roles of renal fibroblasts might lead to the development of a new therapeutic approach for kidney diseases. Recent novel technologies such as scRNA-seq have revealed the heterogeneity of renal fibroblasts that had not previously been identified by conventional technologies . The application of these technologies to various clinical renal diseases is expected to further clarify the heterogeneity of renal fibroblasts, which will result in an enhanced understanding of the pathophysiology of kidney diseases and the development of novel treatments.
Conflict of interest
YS is employed by the TMK Project, which is a collaboration between Kyoto University and Mitsubishi Tanabe Pharma. MY receives research grants from Mitsubishi Tanabe Pharma and Boehringer Ingelheim. TY reports no conflicts of interest.
Appendices and nomenclature
|CKD||chronic kidney disease|
|ESRD||end-stage renal disease|
|TLT||tertiary lymphoid tissue|
|p75NTR||p75 neurotrophin receptor|
|P0||myelin protein zero|
|α-SMA||α-smooth muscle actin|
|scRNA-seq||single cell RNA-sequencing|
|MSC||mesenchymal stem cell|
|iDTR||inducible diphtheria toxin receptor|
|UUO||unilateral ureteral obstruction|
|AKI||acute kidney injury|
|TGFβ-1||transforming growth factor β-1|
|PHD||prolyl hydroxylase domain–containing protein|
|SGLT2||sodium-glucose cotransporter 2|
|RALDH2||retinaldehyde dehydrogenase 2|
|RAR||retinoic acids receptor|
|DAMP||damage-associated molecular pattern|
|FDC||follicular dendritic cell|