Kristen in her office

The complex but interesting featured graphic introduces aspects of the innate immune system which is present and active in the kidney and may have a role in stone genesis. Although innate immunity in kidney is a well established area of research, the specific links to plaque and stone formation have not been explored thus far. So, my post is meant to interest people in the possibility and perhaps give rise to some new scientific research.

Toll Like Receptors (TLRs)

The innate immune system is an ancient system of defense against environmental threats that is found in all classes of plant and animal life. Innate immunity provides immediate, non-specific defense against infection and products of cell damage or stress. Toll-like receptors (TLRs), sentinels of the innate immune system, are proteins that recognize structurally conserved molecules derived from microbes or injured cells. Eleven human TLRs have been identified and are expressed either on the cell surface (TLR1/2/4/5/6/10/11) or in intracellular endosomes (TLR3/7/8/9).

Renal TLRs

Several TLRs are normally expressed by renal tubular epithelial cells all along the nephron, and some are known to be up-regulated under conditions of infection or cellular stress, such as TLR4 and TLR11 during urinary tract infection, and TLR2 and TLR4 with ischemia or sepsis.  Renal ischemia/reperfusion injury leads to marked enhancement of both TLR2 and TLR4 levels, mainly in epithelial cells of the distal tubules, collecting ducts, and thin limbs of Henle’s loops.

Renal TLRs and Inflammation

Binding of TLR4 by any of a number of ligands triggers the expression of pro-inflammatory cytokines and chemokines that mediate innate immunity. Ligands of TLR4 include pathogen associated molecular pattern molecules (PAMPs), such as bacterial LPS, and endogenous cell damage associated molecular pattern molecules (DAMPs), such as various heat shock proteins, hyaluronic acid fragments, S100A9 (calgranulin B) and Tamm Horsfall Protein (uromodulin).

Tubule lumen crystals and TLRs

Calcium phosphate crystals have also been found to be a ligand of TLR4. If, as John Asplin’s work suggests, calcium phosphate crystals are prone to form in the thin limbs of the loops of Henle, the interaction of these crystals with TLR4 on tubular epithelial cells may set off an innate immune response in this location.

How TLRs may Cause Plaque Crystals to Form 

TLRs may cause local phosphate release

Extracellular ATP is another pro-inflammatory local signaling molecule that is released from renal epithelial cells that are stressed or injured. A counter-inflammatory feature of the innate immune response is that, as a consequence of TLR4 ligation, ectonucleotidases CD39 and CD73 are upregulated in order to limit inflammatory signaling by ATP: extracellular ATP is rapidly converted to AMP by ecto-nucleoside-triphosphate-diphosphohydrolase1 (CD39) and AMP is converted to adenosine via phosphohydrolysis by the ecto-5′-nucleotidase (CD73). This leads to release of inorganic phosphate. For every ATP molecule that is converted to adenosine by CD39 and CD73, three inorganic phosphate (Pi) molecules are generated.

TLRs may increase phosphate molarity around thin limbs and Bellini ducts

In rats, CD73 is expressed in the peritubular capillaries and interstitial fibroblasts in the peritubular space, as well as the inner medullary collecting ducts; CD39 is expressed in the ascending thin limbs of the loops of Henle and in ducts of Bellini. These are strikingly similar locations to where hydroxyapatite crystal deposits, the start of Randall’s plaque, have been found in the renal papillae of idiopathic CaOx stone formers. Since three inorganic phosphate (Pi) molecules are generated for every ATP molecule that is converted to adenosine by CD39 and CD73, upregulation of these enzymes and plentiful ATP substrate from stressed cells may lead to excess Pi in the interstitium relative to pyrophosphate (PPi), a potent inhibitor of hydroxyapatite deposition normally found in the kidney. A reduced interstitial PPi/Pi ratio, along with excess calcium in the renal interstitium from vas washdown may promote hydroxyapatite formation and deposition of Randall’s plaque.

TLRs may increase urothelial permeability

According to the plaque model, CaOx stone growth would follow deposition of Randall’s plaque and exposure of plaque to the urine. Plaque is separated from the urinary space by a thin covering of renal papillary epithelial cells. Activation of innate immunity by TLR ligation induces the expression of three cytokine classes: IL-1β, interferons (IFN-α and IFN-β) and NF-kB-dependent cytokines and chemokines, such as IL-6 and TNFα. Epithelial cell barrier function is maintained by intercellular tight junctions (TJ) that seal the space between adjacent cells; cytokines such as TNFα and interferons perturb TJ formation by inducing actin remodeling and changes in TJ structure, allowing increased paracellular permeability. TNFα may promote leakage of the papillary surface epithelium allowing urinary components to enter the interstitium, leading to overgrowth of CaOx on plaque.

Evidence for TLR activation in stone disease

Increases in ‘Crystal Inhibitory Molecules’

We have evidence that some proteins whose expression is known to be induced by innate immune activation are altered in the kidneys or urine of stone formers. Differences in form and abundance of S100A9 and inter-α-trypsin inhibitor (ITI) in urine are associated with calcium stone formation. In addition, osteopontin and heavy chain 3 of ITI (ITIH3) co-localize in the matrix of Randall’s plaque and cells of the thin loops of Henle. All of these proteins are found in the matrix of calcium stones, with S100A9 typically being a major component. Likewise all of these are part of the suite of molecules long studied as modulators of crystallization in urine.

The Special Role of S100A9

S100A9 is of particular interest as an injury response protein that is highly expressed in numerous inflammatory conditions, including giant cell arteritis, cystic fibrosis, rheumatoid arthritis, dermatoses, some malignancies, and autoimmune diseases such as chronic inflammatory lung and bowel diseases. S100A9 is also an avid calcium binding protein that is strongly upregulated in calcifying areas and surrounding extracellular matrix of atherosclerotic plaque. It is expressed in human bone and cartilage cells where it may play a role in matrix calcification. S100A9 is elevated in synovial fluid from patients with arthritis and may modulate cartilage calcification. S100A9 is also a major component, along with hydroxyapatite, of prostatic calculi (corpora amylacea) which are linked to aging and prostate inflammation. In addition, S100A9 is increased in the brains of patients with Alzheimer’s disease and is associated with amyloid plaque formation.

S100A9 is a ligand of TLR4 (DAMP) and as such may induce a “damage chain reaction” in which proinflammatory mediators are upregulated, triggering further tissue damage and resulting in a state of chronic innate immune activation. Progressive cell damage produces more endogenous TLR agonists (DAMPs) which may serve to perpetuate the innate immune response, and consequently plaque and stone formation, via an autoamplification loop of injury and inflammation. For example, tubular injury allows uromodulin leakage into the interstitial compartment where it becomes a DAMP that activates interstitial dendritic cells via TLR4. In the presence of cellular damage that exposes the renal interstitium, uromodulin also becomes an endogenous danger signal that activates the intracellular NLRP3 inflammasome complex, as do CaOx crystals themselves.

TLRs may promote renal fibrosis in stone formers

Cellular release of IL-1β and IL-18 due to NLRP3 activation initiates downstream pro-fibrotic and inflammatory effects, as well as apoptosis or lytic cell death. Uric acid crystals are similarly activators of the NLRP3 inflammasome. DAMPs additionally trigger re-epithelialization upon kidney injury and contribute to epithelial-mesenchymal transition and possibly to myofibroblast differentiation and proliferation. Thus, DAMPs drive not only immune-mediated injury but also kidney cell regeneration and renal scarring, potentially leading to theinterstitial inflammation and fibrosis seen in the kidneys of some stone formers with advanced disease.


  1. Kristin Bergsland

    Thank you for your comments concerning C-reactive protein (CRP) and for pointing out your very interesting paper. The relationship between higher serum levels of CRP and increased prevalence of kidney stones is indeed intriguing. CRP is a fairly small protein with a molecular weight of about 25 kDa which should allow it to be easily filtered through the glomerulus. Although the majority of filtered proteins are reabsorbed in the proximal tubule, some low molecular weight proteins make their way into the tubular fluid. Those that are ligands of TLRs could potentially activate innate immunity along the nephron. It’s possible that increased CRP in the filtrate, reflecting higher serum levels, might lead to activation of TLR4 and promotion of plaque and stone formation in a similar fashion to that outlined in the above post. Likewise, reduced proximal tubular reabsorption of proteins due to injury or other dysfunction could lead to increased concentrations of TLR ligands in downstream tubular fluid. In any case, this is a ripe area for future research.
    Thanks, Kristin

  2. Hari Koul, PhD

    Kristin and Fred-
    Here is what i am working on. This abstract seas written in Jan 2014 as part of proposed Obrian grant application from UF, that did not get off ground. I believe based on our data that TLR activation may be a central event that drives crystal retention. Importantly TLR4 activation may connect crystal induced calcification process to the infection induced calcification process as we have hypothesized earlier this year.
    Here is the abstract of our proposed work:

    Elevated levels of oxalate as well as urinary tract infections have independently been associated with subsets of idiopathic stone formers. However, precise mechanisms by which moderately elevated levels of oxalate and or renal tubular infections promote kidney stone formation are not understood. In addition interplay between moderately elevated oxalate and urinary tract infections in driving stone disease has not ever been studies. Idiopathic stone formers often present with mixed type stones. The stones contain calcium oxalate as well as calcium phosphate deposits. There is a large body of literature that supports the notion of presence of Randall’s plaques (sub-epithelial Calcium Phosphate precipitates) associated with deposits of Calcium oxalate. While tubular precipitation of calcium oxalate as well as calcium phosphate can be explained by urinary precipitations as a result of elevated levels of calcium, phosphate and /or oxalate, the exact mechanisms that govern the formation of Randall’s plaques is not clear. Our studies and those of others over last two decades have demonstrated that oxalate exposure to the renal epithelial cells results in a program of events including cell damage, re-initiation of DNA synthesis as well as cell death. We have also shown for the first time that oxalate interactions result in activation of p38 MAP/ stress kinase signal transduction pathway and activation of NF-KB transcription factor. We discovered expression of IL-6 in renal epithelial cells in response to oxalate and calcium oxalate crystals. The response to oxalate was similar to LPS , a bacterial wall component that has been shown to activate expression of IL-6. Our pioneering studies using global gene expression profiling has further led us to uncover new and pathways that are activated in response to renal epithelial cells to oxalate. We are the first group to have identified expression of IL2b receptor in renal epithelial cells in response to oxalate. In preliminary studies we present evidence that suggests activation of TLR4 as a key driver of altered signaling cascades in response to oxalate. We also present preliminary evidence that oxalate as well as LPS do indeed activate TLR 4 pathway. These observations form the basis of our central hypothesis to be tested in this application, that “Activation of TLR-4 pathway is critical driver of renal epithelial cell response to elevated oxalate and LPS.” This hypothesis will be tested in three specific aims.
    In the first specific aim we will test the hypothesis that, “Moderately elevated levels of oxalate and LPS act synergistically in activation of TLR-4” In the second specific aim we will test the hypothesis that “oxalate induced activation of p38 MAP kinase and NF-KB activation is dependent on the activation of TLR-4 pathway”. We will also evaluate relative contribution of elevated levels of oxalate and LPS in activation of these cascades. In the third specific aim we will test the hypothesis that “moderately elevated levels of oxalate and LPS co-operate to modulate renal epithelial cell phenotype to promote calcium precipitation”. Finally in the fourth specific aim we will examine these events in vivo.

    • Kristin Bergsland

      Thank you for the thoughtful comments. I totally agree that TLR4 may connect crystal-induced and infection-induced calcification processes. With regard to LPS activation of TLR4 in the kidney, it is possible that LPS may be present not only in the distal nephron, as a result of ascending infection, but also in the proximal tubule as a result of descending TLR4 ligands from the bloodstream. It is known that low levels of LPS (1-10 pg/ml) thought to be derived from gut bacteria are present in the blood of healthy people. Moderate, subclinical elevation of serum LPS (50-100 pg/ml) is associated with insulin resistance, obesity and chronic low grade inflammation; these are all conditions that are also associated with increased risk of stones. LPS readily passes through the glomerular barrier where it can be taken up by proximal tubular cells in a TLR4-dependent fashion, leading to oxidative stress and innate immune activation. Products of proximal tubule cell stress and damage (DAMPs) may then enter the tubular fluid and be carried downstream to activate innate immunity in the region of the nephron where plaque formation is known to occur.

      Your suggestion of the involvement of oxalate in activation of the TLR4 pathway is intriguing. If true, elevated oxalate concentrations in tubular fluid might stimulate plaque and stone formation not only by promoting calcium oxalate crystallization but also by activation of TLR4 along the nephron in a similar way to that outlined above. If, as you hypothesize, oxalate and LPS act synergistically in the activation of TLR4, it will serve to exacerbate this effect. Interestingly, IL-6, a pro-inflammatory cytokine produced in response to TLR activation, has been found to be significantly increased in the urine of patients with upper urinary tract calcium stones compared with healthy controls. This seems in accordance with your findings of elevated IL-6 production in renal epithelial cells in vitro in response to oxalate. In addition, you have shown that oxalate provokes expression of HSP70 in proximal tubular epithelial cells (LLC-PK1). HSP70 is an endogenous ligand of TLR4 (DAMP) which could likewise lead to further innate immune activation along the nephron. Clearly the intersection of chemistry/crystallization processes and innate immunity in the kidney is a fertile area for future research.
      Thanks, Kristin


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