The Role of Xiaobo Li's Rush University Patents in Understanding and Treating Glomerular Injury

Diabetic glomerular injury stands as a major complication of diabetes mellitus, recognized as the primary cause of end-stage renal disease (ESRD). The health and proper function of podocytes are essential for the glomerulus of the kidney to function properly. Podocyte injury or loss leads to increased proteinuria and kidney dysfunction, a common characteristic in various glomerulopathies. Therefore, understanding the mechanisms of pathways that protect podocytes from damage is crucial for the development of future treatments.

The Significance of MicroRNA-146a (miR-146a)

MicroRNA-146a (miR-146a) acts as a negative regulator of inflammation and shows high expression in myeloid cells and podocytes. Prior research indicates that miR-146a levels are significantly reduced in the glomeruli of patients with diabetic nephropathy (DN). To further investigate this, mice with selective deletion of miR-146a in podocytes were generated and used in models of glomerular injury.

Experimental Evidence: Protecting Podocytes from Damage

Induction of glomerular injury in C57BL/6 wildtype mice (WT) and podocyte-specific miR-146a knockout (Pod-miR146a-/-) animals through administration of low-dose lipopolysaccharide (LPS) or nephrotoxic serum (NTS) resulted in increased proteinuria in the knockout mice. This suggests that podocyte-expressed miR-146a protects these cells, and therefore the glomeruli, from damage.

Induction of hyperglycemia using streptozotocin (STZ) also resulted in an accelerated development of glomerulopathy and a rapid increase in proteinuria in the knockout animals, as compared to the WT animals. This further confirms the protective role of podocyte-expressed miR-146a.

The direct miR-146a target, ErbB4, was significantly upregulated in the diseased glomeruli. Erlotinib, an ErbB4 and EGFR inhibitor, reduced its upregulation and the proteinuria in treated animals. Primary miR146-/- podocytes from these animals also showed a basally upregulated TGFβ-Smad3 signaling in vitro.

The Clinical Context of Diabetic Nephropathy

Diabetes mellitus affects a significant portion of the American population, remaining one of the most common medical conditions. Progressive glomerular kidney injury due to diabetes mellitus is the leading cause of end-stage renal disease (ESRD) in the US. Diabetic glomerulopathy is characterized by loss of glomerular podocytes, glomerular basement membrane (GBM) thickening, segmental glomerulosclerosis, and mesangial expansion.

The Vital Role of Podocytes

Podocytes are specialized cells located in the Bowman’s capsule of the glomerulus, essential for the formation and function of the urinary filtration barrier in the kidney. These cells are vital to a healthy glomerulus and normal kidney function. Diabetes results in significant podocyte injury, although the exact molecular mechanisms are unclear. While some disease-associated pathways have been elucidated in recent studies, the mechanisms that lead to podocyte damage during diabetic nephropathy (DN) pathogenesis are yet to be fully understood.

MicroRNAs (miRNAs) as Regulators

MicroRNAs (miRNAs) are a family of small, non-coding regulatory RNAs, approximately 18-22 nucleotides (nt) in length. Among their key functions, they regulate post-transcriptional expression of their target genes by promoting messenger RNA (mRNA) degradation or suppressing mRNA translation into functional proteins by binding the 3’ untranslated region (UTR) of target mRNAs in a sequence-dependent manner. Thus, miRNAs play a vital role in regulating cell biological functions.

Like protein expressing mRNAs, the expression of miRNAs is also regulated in a tissue-specific fashion. Different miRNAs are expressed in all stages of kidney development and are also differentially regulated in the glomeruli in response to various external stimuli, indicating their involvement in disease pathogenesis. miRNAs are essential in podocyte homeostasis, as conditional deletion of miRNA processing enzymes Drosha and Dicer results in significant proteinuria and accelerated glomerular injury. Similarly, deletion of specific miRNAs in podocytes results in increased proteinuria and glomerulosclerosis.

miR-146a as a Key Regulator in Podocytes

MicroRNA-146a (miR-146a) is a negative regulator of the pro-inflammatory signaling in myeloid cells and thus a key molecular brake on the inflammatory innate immune cell responses. It also modulates key adaptive immune cell functions and plays an important role in hematopoiesis and cancer cell proliferation, via targeting a different set of genes. miR146a is also highly expressed in the podocytes and in all other in types of cells in the glomeruli, suggesting that it has important homeostatic and regulatory roles in podocytes, including regulating kidney function during diabetic injury.

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Myeloid cell-expressed miR-146a was recently shown to increase in expression in murine DN, where it plays an anti-inflammatory role by suppressing proinflammatory cytokines in macrophages. Conversely, glomerular miR-146a levels were dramatically reduced in the kidney sections of diabetic patients, and the level of miR-146a expression in the kidneys inversely correlated with proteinuria in the patients, suggesting that podocytic miR-146a plays a protective role in DN. A recent study with 460 subjects (300 cases and 160 controls) also confirmed that there exits an inverse association of blood miR-146a levels with diabetes and its complications. Furthermore, miR-146a levels were found to be down-modulated in the kidneys of diabetic rats and mice, suggesting that it plays a reno-protective role.

Podocyte-Specific miR-146a Knockout Mice

The absence of miR-146a in the global miR-146a knockout animals (miR-146a-/-) increased susceptibility of these animals to diabetic kidney disease, via hyperglycemia-induced podocyte damage. To address this, podocyte-specific miR-146a-/- animals (Pod-miR146a-/-) were generated to study the functional consequences of the absence of miR-146a specifically in the glomerular podocytes. The Pod-miR146a-/- mice mimic the diabetic glomerular injury observed in the global miR-146a-/- animals. Additionally, given that miR-146a directly targets tyrosine receptor kinase ErbB4, the absence of miR146a results in increased ErbB4 expression and signaling, thus driving a podocyte-damaging feed-forward loop.

Healthy podocytes abundantly express miR-146a, where it plays an important role in cellular health and function. miR-146a levels significantly decreased in the glomeruli of diabetic patients and that the decrease was associated with progressively increasing kidney damage. Furthermore, using animals with global deletion of miR-146a (miR146a-/-), it was also shown that the absence of miR-146a accelerated the progression of diabetic nephropathy (DN) in the knockout animals.

The Hypothesis: Podocyte-Expressed miR-146a

Given previous data with DN patient biopsies and the DN models in miR-146a-/- mice, it was hypothesized that the podocyte-expressed miR-146a plays an essential role in protecting glomeruli from injury. To unambiguously explore this role of miR-146a, a novel, podocyte-specific miR146a deleted transgenic mouse (referred to as Pod-miR146a-/-) was developed using published protocols.

Briefly, C57BL/6 miR146afl/fl mice containing floxed sites around the miR146a exon were first crossed with Flp-recombinase expressing mice to remove the selection cassette containing LacZ and neomycin. Next, homozygous progeny were crossed with Podocin-Cre recombinase-expressing mice, which recognize the loxP sites surrounding the miR146a gene and only cut these sites in cells under the control of the Podocin promoter (which is highly and selectively in podocytes), thus selectively deleting miR146a specifically in podocytes to obtain Pod-miR146a-/- mice. The resulting mice were backcrossed >6 times before use in experiments. Quantitative RT-PCR (qRT-PCR) based analyses of isolated podocytes showed an almost complete loss of miR-146a expression, as compared to miR-146a levels in cells from the wild type C57BL/6 wild-type (WT) littermates, whereas miR-146a expression in spleen and whole kidney showed no significant difference, as compared to the WT animals.

LPS-Induced Glomerular Injury

To evaluate the effect of podocyte-specific miR146a deletion in models of glomerular injury, its role in low-dose lipopolysaccharide (LPS) induced glomerular injury model of focal segmental glomerulosclerosis (FSGS) was examined. Expectedly, LPS administration into WT mice resulted in a strong induction of albuminuria after 24 h compared with vehicle controls. LPS administration into the Pod-miR146a-/- animals produced a significantly higher rise in albuminuria as compared to the LPS-treated WT animals, suggesting the lack of miR-146a in podocytes exacerbates glomerular injury. Histochemical analyses confirmed significant glomerular damage in the LPS-treated groups compared to controls.

Anti-GBM Nephritis Model

The role of miR146a in glomerular injury was further investigated using an established model of anti-glomerular basement membrane (anti-GBM) nephritis. Administration of nephrotoxic serum (NTS) into WT and Pod-miR146a-/- mice resulted in progressively increased albuminuria, with the Pod-miR146a-/- animals showing significantly worse disease and peak proteinuria at 4 weeks post NTS administration. This suggests that miR146a plays an important role in protecting glomeruli from chronic injury. Histochemical analyses further confirmed significantly higher glomerular damage, including increased mesangial matrix expansion, in the NTS-treated Pod-miR146a-/- mice.

STZ-Induced Hyperglycemia

To further investigate the effect of podocyte-specific miR146a deletion on glomerular function in vivo, hyperglycemia was induced in WT or Pod-miR146a-/- animals using published STZ protocols. STZ treatment resulted in induction of hyperglycemia and body weight decline in WT animals and to a similar extent in the Pod-miR146a-/- mice, confirming recent findings with the global miR-146a-/- mice.

ErbB4 and Erlotinib

ErbB4 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4, also known as HER4) is a tyrosine kinase receptor that is a member of the epidermal growth factor receptor (EGFR, also known as ErbB) family of receptors. ErbB4 often heterodimerizes with EGFR to form a functional receptor. ErbB4 mRNA is a direct molecular target of miR-146a, and binding of miR-146a to ErbB4 mRNA targets ErbB4 for degradation.

Previously, ErbB4/EGFR expression and signaling was observed to be upregulated in the diabetic miR-146a-/- animals and that blockade of this pathway with clinically available ErbB4/EGFR inhibitor erlotinib was therapeutic, and erlotinib treatment reduced ErbB4 and EGFR expression and signaling. To determine if this pathway was also therapeutic in the context of podocyte-specific miR-146a deletion, erlotinib was also administered to a group of diabetic WT and Pod-miR146a-/- animals. Results show that erlotinib treatment significantly reduced the level of albuminuria in both the diabetic WT and Pod-miR146a-/- animals, without affecting the increased hyperglycemia or decreased body weight, as expected. This suggests that podocytic miR-146a protects cells via suppression of ErbB4.

Genetic Variations in ITGAM and Systemic Lupus Erythematosus (SLE)

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Three nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-β, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b.

Systemic lupus erythematosus (SLE, lupus) is a debilitating autoimmune disease that is characterized by hyperactive immune cells, serum autoantibodies, immune complex deposition, multiorgan damage, and accelerated vascular disease. Pathways downstream of various pattern recognition receptors - in particular, Toll-like receptors (TLRs) - are central to the aberrant immune responses contributing to SLE and result in elevated levels of inflammatory cytokines, such as type I interferons (IFN-I), IL-1β, IL-6, TNF-α, and IL-17, in patients. Elevated levels of IFN-I in circulation are a heritable risk factor for SLE and play a pathogenic role. Although the underlying mechanisms influencing IFN-I levels in SLE subjects and conferring a strong predisposition to disease remain to be fully characterized, genetic variations, in combination with environmental stressors, play a key role.

Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) in a number of immune response genes as key contributors to disease activity, and SNPs in ITGAM (coding for CD11b or αM, the α chain of the β2 leukocytic integrin heterodimer CD11b/CD18) show highly significant correlation with SLE. Three ITGAM coding region SNPs, which result in missense mutations P1146S (a C>T substitution), R77H (a G>A substitution), and A858V (a C>T substitution), respectively, in the protein, have strong correlation with the incidence of SLE and with SLE subphenotypes including lupus nephritis, discoid rash, and immunologic manifestations. How these nonsynonymous mutations confer SLE risk is not clear, but they appear to reduce CD11b function, including integrin activation, ligand binding and cell adhesion, phagocytosis, and catch-bond formation.

Recent studies also show that CD11b acts as a negative regulator of TLR signaling pathways and of B cell autoreactivity, implying that the ITGAM SNPs found in SLE patients may affect disease activity via a reduction in CD11b’s normal, antiinflammatory signaling activities in leukocytes, although the exact mechanism behind how these variants contribute to autoimmunity is unclear. In murine systems, while CD11b deficiency was shown to reduce neutrophil accumulation and glomerular injury in an acute model, it has also been shown to increase susceptibility to chronic inflammatory and autoimmune diseases, including increased tissue infiltration of leukocytes and immune-mediated injury in lupus-prone mice. These data further suggest that CD11b plays a protective role in SLE.

ITGAM SNPs and Elevated IFN-I Activity

SLE patients carrying SNPs in the ITGAM gene present with elevated IFN-I activity, molecularly linking reduced CD11b function with increased disease risk. Ex vivo, cells homozygous for ITGAM SNPs showed a basal increase in IFNB and IRF7 expression and a decreased level of nuclear FOXO3, which regulates their expression, suggesting reduced suppression of TLR signaling at a basal level. Pharmacologic activation of CD11b, protein product of ITGAM, reduced IFN-I responses ex vivo and in animals via suppression of TLR-dependent FOXO3/IRF7 pathway in a CD11b-dependent fashion. It also similarly protected lupus-prone mice from end-organ injury.

The ITGAM risk alleles showed strong association with high IFN-I serum activity in patients. Furthermore, analysis showed that the SNPs rs1143678 and rs1143683 were in complete linkage disequilibrium, forming a haplotype. Regression analysis demonstrated that alleles in rs1143679 and rs1143678/rs1143683 showed independent evidence for association with elevated IFN-I serum activity. Surprisingly, a subset of these subjects carried a haplotype containing the common SNP in rs1143679 (G) along with the rare SNP in rs1143683 (T). This GT haplotype was also associated with significantly elevated IFN-I activity.

Disease Activity and ITGAM Risk Allele Carriers

To examine whether there was increased disease activity or flare in the ITGAM risk allele carriers at the time of blood draw that might account for this association, their SLE Disease Activity Index (SLEDAI) scores were examined. The average SLEDAI score in the overall cohort was 3.16, indicating mild to moderate average disease activity, typical of an outpatient SLE cohort. While there seems to be a trend toward higher disease activity, no significant increase in SLEDAI in the ITGAM risk allele carriers versus noncarriers was found. Furthermore, it was examined in a multivariate model, by performing a logistic regression analysis to assess the impact of SLEDAI score on the relationship between the ITGAM SNPs and IFN-I levels. SNPs were entered as predictor variables, with IFN as the outcome, and the model was assessed with and without inclusion of SLEDAI score.

These analyses suggest a direct link between reduced CD11b function as a result of these SNPs and the increased IFN-I activity in SLE. CD11b/CD18 ligation and clustering mediate proinflammatory signaling in leukocytes, including synergistic potentiation of signaling by other receptors. Conversely, they have also been shown to negatively regulate signaling by TLRs, TNF receptors, and cytokine receptors, which could suppress downstream IFN-I pathways.

Pharmacologic Activation of CD11b

To test whether pharmacologic activation of CD11b could suppress TLR-dependent responses, and thus be a therapeutic strategy in SLE, our recently discovered small-molecule CD11b agonist LA1 was used as a tool. LA1 binds to the CD11bA/I domain (CD11bA) in an allosteric pocket, enhancing ligand binding. Molecular dynamics and isothermal calorimetry studies with CD11bA and fluid shear stress studies with cell surface-expressed full-length integrin CD11b/CD18 further established that LA1 binding promoted partial CD11bA activation, inducing an intermediate conformation in CD11bA and no large conformational changes in the integrin heterodimer, defining this as its preferred binding mode.

Given that the SLE-associated CD11b mutations are located outside of the ligand-binding αA domain, it is unclear whether they would affect LA1 binding to the αA domain, and thus affect LA1’s activity. To assess, cells stably expressing either full-length WT integrin CD11b (and its partner CD18) or the CD11b R77H mutant were generated and used them in adhesion assay. The results show that both cell types displayed a similar level of LA1-mediated adhesion to immobilized Fg, with similar EC50 values, suggesting that LA1 is able to activate the WT and the mutant integrin equally well and its binding is unaffected by the ITGAM SNP. The results also corroborate a recently published study, which also showed that the functional defects in CD11b R77H are rescued by integrin activation.

LA1 Suppression of Proinflammatory Signaling

Primary murine macrophages and neutrophils were treated with LPS in the absence or presence of LA1, and secretion of IL-1β, IL-6, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) in cellular supernatants was measured. LA1 significantly suppressed levels of all 4 in macrophages and neutrophils in a dose-dependent manner. Similarly, TNF-α-stimulated IL-6 production was also significantly reduced. LA1 treatment also suppressed levels of IL-6 and TNF-α in cell culture supernatants of human macrophages stimulated with the TLR agonist LPS or R848, further validating the role of LA1-mediated integrin activation as suppressor of proinflammatory signaling by heterologous receptors in leukocytes.

As TLR activation also induces IFN-I and downstream IFN-I signaling pathways, the effects of TLR stimulation on IFN-I generation in vivo were tested. LPS administration significantly upregulated IFN-β levels, with the level of increase significantly higher in the CD11b-/- animals as compared with the WT animals, suggesting that CD11b activation-dependent modulation of TLR-signaling extends to IFN-I pathways. Importantly, CD11b activation with LA1 significantly suppressed this upregulation of IFN-β in WT mice, but not in the mice lacking CD11b (CD11b-/-), suggesting that the in vivo effects of LA1 here are mediated via CD11b. In vitro, LPS treatment also resulted in significantly increased production of IFN-β by CD11b-/- macrophages, as compared with the WT cells, in accordance with the published studies. Furthermore, while LA1 significantly reduced the LPS-mediated upregulation in IFN-β levels in the cultured macrophages from WT animals, it failed to suppress IFN-β upregulation in the CD11b-/- cells.

In Vivo Effects of CD11b Activation with LA1

To study the effects of CD11b activation with LA1 on TLR-stimulated cytokine generation in vivo, WT C57BL/6 mice were subjected to cecal ligation and puncture (CLP), a model of polymicrobial sepsis, and treated them with LA1 or vehicle control. Among the control, vehicle-treated mice, 75% died within 120 hours, and more than 90% of them died within 240 hours. Surprisingly, more than 60% of LA1-treated mice were alive for 120 hours, and 50% of them survived more than 240 hours. Vehicle-treated mice showed high serum levels of the 3 hallmark cytokines (IL-1β, IL-6, and TNF-α) that play a significant role in cytokine storm-mediated septic shock, whereas LA1-treated animals showed a significant reduction in these cytokines. Significantly, serum bacterial count 24 hours after CLP showed few remaining bacteria in LA1-treated mice as compared with high levels in the vehicle-treated group, which suggests that LA1 treatment also suppresses bacterial load and correlates with previous data that LA1 enhances phagocytosis.

Viral infections also lead to exacerbated leukocyte activation and cytokine storm, which prospectively predicts morbidity and mortality in patients. To determine whether LA1-mediated integrin activation in leukocytes would similarly suppress excessive immune response upon severe viral infection, C57BL/6 mice were infected via intranasally.

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