Biology of Renal Recovery: Molecules, Mechanisms and Pathways

Abstract

Acute kidney injury contributes to progressive kidney disease. Although significant advances have been made in the understanding of mechanisms of acute kidney injury, less is known about the biological basis that links the initial injury to progressive interstitial fibrosis, tubular dysfunction and capillary rarefaction. The round table discussion focused on mechanisms of renal recovery and fibrosis following acute kidney injury. The knowledge gained by understanding these pathways will serve to identify novel therapeutic targets in the future.

Acute kidney injury (AKI) results in temporary loss of kidney function. In some cases, this initial injury leads to progressive kidney disease and end stage renal disease (ESRD). However, the kidney also has a remarkable ability to undergo reparative processes leading to regeneration and recovery following AKI. Over the last two decades there has been great progress to identify mechanisms that lead to regenerative processes. This review will cover the mechanisms of recovery from AKI incorporating the discussion at the Round Table in San Diego at the CRRT 2014 and current literature regarding the source of cells that contribute to the regenerative process including stem cells, epithelial cells, pericytes, and bone marrow-derived cells. This review will also cover the maladaptive process of tubulointerstitial fibrosis, which is the deposition of extracellular matrix components that lead to loss of kidney function.

Aging and Senescence

With age, there is a decreased capacity for repair, and chief among the biological processes that account for aging is somatic senescence. Senescence refers to an irreversible growth arrest with viable cells lacking mitogenic potential, so cells do not die but their ability to grow is impaired [1]. Telomeres are nucleoproteins consisting of repetitive DNA sequences and specific proteins that are located at the end of all eukaryotic chromosomes. Telomeres control chromosome stability, genetic integrity, and cell viability, and telomerase enzymes are thought to play a role in cell senescence [2]. Other factors such as oxidative stress, DNA damage and mitochondrial injury contribute to non-telomerase dependent cell senescence. These factors contribute to the increase in incidence of AKI and decreased capacity for repair in the aging population [1,3].

Cell Injury

In most animal models of ischemia-reperfusion injury (IRI), the S3 segment of the proximal tubule is most affected and tubule cells undergo apoptosis and necrosis [4]. Hypoxia is an early stimulus that inactivates prolyl-hydroxylase thus inhibiting hypoxia inducible factor-1 alpha (HIF-1α) degradation. HIF-1α then translocates to the nucleus and binds to HIF-1β leading to transcription of HIF target genes resulting in angiogenesis, apoptosis, cell proliferation, cell survival, and glucose metabolism, factors that lead to both oxygen delivery and adaptation to oxygen deprivation [5]. Heme oxygenase-1 (HO-1) and galectin 1 are important HIF-1α target genes. HO-1 is known to suppress inflammation and AKI [6,7], whereas galectin-1 is thought to suppress inflammation and may be cardioprotective in myocardial infarction [8]. HIF prolyl-hydroxylase inhibitors are currently in development and some are in use in clinical trials (ClinicalTrials.gov).

Mitochondria exist in dynamic equilibrium and are a major source of energy in proximal tubule cells, thus they play a central role in cell survival, injury and death [9]. They undergo constant fusion and fission to maintain the health of cells. Mitochondrial fusion is mediated by mitofusin 1 (Mfn1), Mfn2 and optic atrophy 1 (OPA1), and fission is mediated by dynamin-related protein 1 (Drp1) and mitochondrial fission 1 (Fis1). Alterations in these proteins lead to impaired mitochondrial function and cell death through reduced ATP, release of pro-apoptotic proteins and increased oxidants. Following mitochondrial injury, mitochondria are sequestered by a process referred to as mitophagy. The engulfment of injured mitochondria may prevent cell death [9] and regulation of mitophagy may be a novel strategy to prevent cellular death following IRI. Rapamycin attenuates cisplatin nephrotoxicity, in part, through enhanced mitophagy, whereas chloroquine blocks mitophagy and exacerbates AKI [10]. The synthesis of new mitochondria, referred to as mitochondrial biogenesis, may be an important mechanism that assists in renal recovery. The peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC1α), a transcriptional co-activator, binds to several transcription factors that regulate mitochondrial biogenesis. In sepsis, deficiency of proximal tubule-specific PGC-1α leads to more sustained loss in GFR compared to wild type controls [11], and the sirtuin 1 (SIRT1) activator induces deacetylation of PGC-1α, a marker of activation and enhanced recovery of mitochondrial biogenesis and renal recovery [12].

Immune Cells, Inflammation and Regeneration

Following injury, processes are initiated that lead to regeneration of the tubule epithelium, or under pathological circumstances, lead to fibrosis in which normal parenchymal cells are replaced with connective tissue. Following initial injury acute inflammatory infiltration mediates damage within the kidney. Dendritic cells (DCs) are abundant in the kidney interstitial compartment and interact with exogenous molecules (pathogen-associated molecular patterns, PAMPS) released from pathogens, endogenous molecules (danger-associated molecular patterns DAMPS) released from injured cells, and with resident or infiltrating immune cells including lymphocytes, natural killer T cells (NKT cells), epithelial cells and fibroblasts [13]. Once activated DCs induce injury either directly or through inflammatory signals. Major cell types that contribute to the initial inflammation after AKI are inflammatory monocytes, neutrophils, lymphocytes and NKT cells [14–16].

Monocytes are thought to survey the status of endothelial cells in tissues such as the kidney, and when the endothelium is damaged, they recruit neutrophils to elicit inflammation [17]. Inflammatory monocytes then help clear away apoptotic neutrophils and endothelial cells after injury. Thus monocytes play a prominent role in renal injury as well as repair.

Several cell types regulate regeneration after injury. Monocytes and macrophages orchestrate multiple phases of tissue repair. Their exact role is somewhat obscured by the heterogeneity within monocyte/macrophages populations, perhaps due to phenotypic changes in macrophages over time within damaged tissue or to recruitment of unique populations from outside the injury [18]. Changes in phenotype reflect their changing role throughout the repair process, first promoting initial inflammation, thus increasing initial injury which exacerbates scar formation, but also eventually promoting clearance of excess scar tissue. Macrophages were initially seen as either one of two classic phenotypes: M1 or M2, inflammatory and anti-inflammatory, respectively. This dichotomy however does not seem to fully encapsulate the diverse phenotypes of macrophages during wound repair or fibrosis. M2 macrophages have been further subdivided into M2a, M2b, and M2c phenotypes based on their profiles of cytokine production and function [19]. Macrophages show plasticity in response to environmental signals and therefore their phenotypes may not be terminal.

Macrophages also play a crucial role in regeneration of the kidney after injury and reduction of the scar area. Toll like receptor (TLR) ligands such as lipopolysaccharide (LPS) and cytokines such as IFN-γ induce monocytes to differentiate into M1 macrophages. M1 macrophages promote a pro-inflammatory environment within the kidney through production of TNF-α, which results in apoptosis of endothelial cells within the kidney. These macrophages can be identified by cell surface expression of CCR2. Blockade of CCR2 reduces initial renal inflammation and injury [20]. Clodronate liposomes specifically deplete M1 macrophages and decrease injury [21]. M1 macrophages also play a role in increasing in fibrosis in mice [22], however, it remains to be determined if they exacerbate the initial inflammatory injury or if they inhibit repair.

While typical inflammatory cytokines, PAMPS and DAMPS lead to the classical activated macrophage phenotype, different combinations of cytokines and immune activators induce the alternative phenotypes of macrophages. M2b macrophages produce IL-10 and TGF-β that promote an anti-inflammatory environment. IL-10 and PDGF produced by M2b macrophages stimulate the differentiation of fibroblasts into myofibroblasts, which lay down extracellular matrix (ECM) after tissue destruction. M2c macrophage phenotypes help turn over the ECM thus decreasing the fibrotic area. They also may serve to stimulate the proliferation of renal endothelial cells. This may be due to the ability of macrophages to produce cytokines that promote tubular regeneration [23].

Orchestrating the process of regeneration after AKI is an assortment of soluble molecules such as cytokines, growth factors and peptide molecules such as endothelin. Administration of recombinant EGF to rats after bilateral IRI increased proliferation of tubules [24]. Similarly, studies have shown that growth factors such as PDGF, IGF-1, and HGF are induced within the kidney after injury. Treatment with these molecules increased the proliferation and regeneration of tubule cells after AKI [25]. IL-10 has been shown to be beneficial in decreasing the initial inflammatory damage after both ischemic and cisplatin-induced renal injury [26]. TGF-β potentially inhibits the inflammatory response to AKI and stimulates the regrowth of tubules after injury [27]. Endothelin 1 acts through endothelin type (ET) A (ETA) or ETB receptors. Post ischemic blockade of ETA unlike blockade of ETB decreased tubule/microvascular injury and preserved renal mass. These results suggested that ET-1 acting on ETA may block AKI progression to CKD [28].

Regeneration vs. fibrosis

Following AKI, regenerating epithelial cells are likely derived from endogenous surviving epithelial cells or intrarenal stem cells. Increasingly, it is thought that engraftment of bone marrow-derived cells contribute to a small degree in regenerating kidneys following AKI, suggesting that bone marrow-derived cells do not directly repopulate tubules following injury. On the other hand mesenchymal stem cells home to the site of injury and appear to exert indirect effects to repair injured epithelium through paracrine mechanisms, cell-to-cell communication through soluble factors or microvesicles carrying genetic information [29–31]. Humphreys demonstrated that fully differentiated epithelial cells surviving injury have the capacity to dedifferentiate, proliferate and differentiate into epithelial cells [32]. In addition recent studies have identified a population of vimentin⁺, CD24⁺ and CD33⁺ progenitor tubular cells that are involved in proximal tubule regeneration following AKI [33].

An alternative outcome to kidney regeneration is the maladaptive fibrotic response. Typically myofibroblasts aid in wound contraction and help maintain tissue integrity after damage by depositing extracellular matrix components, such as collagen and fibronectin. However, after severe AKI, this process of matrix deposition is excessive, leading to loss of kidney function and eventually ESRD. The potential cellular origins of myofibroblasts, which has been the focus of much debate, include dedifferentiation of endothelial or epithelial cells (endothelial or epithelial to mesenchymal transition), infiltration of bone marrow-derived populations, or differentiation of local interstitial cells into myofibroblasts. One of the major purported sources of myofibroblasts is the perivascular fibroblasts and pericytes. These cells have a myriad of functions within the kidney such as regulating medullary blood flow. Lin et al. demonstrated in mice that express green fluorescent protein (GFP) under the control of collagen 1 reporter that both pericytes and perivascular fibroblasts express collagen 1 before injury and after UUO [34]. These cells undergo differentiation into myofibroblasts, detected by their upregulation of α-smooth muscle actin (α-SMA). However, bone marrow-derived cells have also been proposed as a source of collagen-producing cells within the kidney and contributed approximately 30% of the α-SMA-positive cells within the injured kidney. More comprehensive work using α-SMA reporter mice has suggested that many of the theorized cellular precursors contribute in varying extents to the population of myofibroblasts within the kidney [35].

Recent studies demonstrate the relationship between cell cycle and fibrosis [36]. Contralateral nephrectomy after unilateral ischemia in mice leads to G2/M arrest in proximal tubule cells resulting in abnormal production of profibrogenic factors. These results suggest that cell cycle arrest may contribute to the pathogenesis of chronic progressive kidney disease and that strategies to maintain proper cell cycle function in epithelial cells following injury may prevent fibrosis and CKD following AKI.

Summary

The Round Table discussion at the CRRT 2014, San Diego provided a wealth of information summarizing the current field in AKI and spanning mechanisms of injury and renal recovery. The biological basis of renal recovery following AKI will likely lead to novel therapeutics designed to target these pathophysiological mechanisms. Combining novel therapeutic and diagnostic strategies - including novel damage biomarkers and physiological biomarkers, such as real time glomerular filtration measurement, renal blood flow, and other imaging modalities - will lead to advances in the care of patients with AKI [37–39].