Sandeep K. Mallipattu

Kruppel-Like factor 15 (KLF15) is a key regulator of podocyte differentiation

Background: Podocyte dedifferentiation is the hallmark of many glomerular kidney diseases. Results: Krüppel-like factor 15 (KLF15) increases podocyte differentiation; KLF15 null mice exhibit more injury in models of kidney disease. Conclusion: KLF15 is a novel transcriptional regulator of podocyte differentiation; a loss of KLF15 increases the susceptibility to kidney injury. Significance: Identification of KLF15 in mediating podocyte differentiation provides new insight into kidney disease. Podocyte injury resulting from a loss of differentiation is the hallmark of many glomerular diseases. We previously showed that retinoic acid (RA) induces podocyte differentiation via stimulation of the cAMP pathway. However, many podocyte maturity markers lack binding sites for RA-response element or cAMP-response element (CREB) in their promoter regions. We hypothesized that transcription factors induced by RA and downstream of CREB mediate podocyte differentiation. We performed microarray gene expression studies in human podocytes treated with and without RA to identify differentially regulated genes. In comparison with known CREB target genes, we identified Krüppel-like factor 15 (KLF15), a kidney-enriched nuclear transcription factor, that has been previously shown to mediate cell differentiation. We confirmed that RA increased KLF15 expression in both murine and human podocytes. Overexpression of KLF15 stimulated expression of differentiation markers in both wild-type and HIV-1-infected podocytes. Also, KLF15 binding to the promoter regions of nephrin and podocin was increased in RA-treated podocytes. Although KLF15−/− mice at base line had minimal phenotype, lipopolysaccharide- or adriamycin-treated KLF15−/− mice had a significant increase in proteinuria and podocyte foot process effacement with a reduction in the expression of podocyte differentiation markers as compared with the wild-type treated mice. Finally, KLF15 expression was reduced in glomeruli isolated from HIV transgenic mice as well as in kidney biopsies from patients with HIV-associated nephropathy and idiopathic focal segmental glomerulosclerosis. These results indicate a critical role of KLF15 in mediating podocyte differentiation and in protecting podocytes against injury.

The changing epidemiology of HIV-related chronic kidney disease in the era of antiretroviral therapy.

The epidemiology of kidney disease in HIV-infected individuals has changed significantly since the introduction of combination antiretroviral therapy (cART) in the mid 1990s. HIV-associated nephropathy (HIVAN), an aggressive form of collapsing focal segmental glomerulosclerosis (FSGS) caused by direct HIV infection of the kidney in a genetically susceptible host, emerged early in the HIV epidemic as a leading cause of end-stage renal disease. With the widespread use of cART, HIVAN is increasingly rare in populations with access to care, and the spectrum of HIV-related chronic kidney disease now reflects the growing burden of comorbid disease in the aging HIV population. Nonetheless, available data suggest that both HIV infection and cART nephrotoxicity continue to contribute to the increased risk of chronic kidney disease in HIV-infected individuals in the United States and Europe. Despite the genetic susceptibility to HIVAN in individuals of West African descent, limited data are available to define the prevalence and spectrum of HIV-related kidney disease in sub-Saharan Africa, which is home to two-thirds of the worlds HIV population. In this mini-review, we characterize the changing epidemiology of HIV-related chronic kidney disease in Western nations and in sub-Saharan Africa.

Expression of HIV transgene aggravates kidney injury in diabetic mice

With the widespread use of combination antiretroviral agents, the incidence of HIV-associated nephropathy has decreased. Currently, HIV-infected patients live much longer and often suffer from comorbidities such as diabetes mellitus. Recent epidemiological studies suggest that concurrent HIV infection and diabetes mellitus may have a synergistic effect on the incidence of chronic kidney disease. To address this, we determined whether HIV-1 transgene expression accelerates diabetic kidney injury using a diabetic HIV-1 transgenic (Tg26) murine model. Diabetes was initially induced with low-dose streptozotocin in both Tg26 and wild-type mice on a C57BL/6 background, which is resistant to classic HIV-associated nephropathy. Although diabetic nephropathy is minimally observed on the C57BL/6 background, diabetic Tg26 mice exhibited a significant increase in glomerular injury compared with nondiabetic Tg26 mice and diabetic wild-type mice. Validation of microarray gene expression analysis from isolated glomeruli showed a significant upregulation of proinflammatory pathways in diabetic Tg26 mice. Thus, our study found that expression of HIV-1 genes aggravates diabetic kidney disease.

Krüppel-like factor 6 regulates mitochondrial function in the kidney

Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Krüppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.

Renoprotective Effect of Combined Inhibition of Angiotensin-Converting Enzyme and Histone Deacetylase

The Connectivity Map database contains microarray signatures of gene expression derived from approximately 6000 experiments that examined the effects of approximately 1300 single drugs on several human cancer cell lines. We used these data to prioritize pairs of drugs expected to reverse the changes in gene expression observed in the kidneys of a mouse model of HIV-associated nephropathy (Tg26 mice). We predicted that the combination of an angiotensin-converting enzyme (ACE) inhibitor and a histone deacetylase inhibitor would maximally reverse the disease-associated expression of genes in the kidneys of these mice. Testing the combination of these inhibitors in Tg26 mice revealed an additive renoprotective effect, as suggested by reduction of proteinuria, improvement of renal function, and attenuation of kidney injury. Furthermore, we observed the predicted treatment-associated changes in the expression of selected genes and pathway components. In summary, these data suggest that the combination of an ACE inhibitor and a histone deacetylase inhibitor could have therapeutic potential for various kidney diseases. In addition, this study provides proof-of-concept that drug-induced expression signatures have potential use in predicting the effects of combination drug therapy.

Deletion of podocyte STAT3 mitigates the entire spectrum of HIV-1-associated nephropathy.

Objective:HIV-1 gene expression in kidney epithelial cells is thought to be responsible for the pathogenesis of HIV-1-associated nephropathy (HIVAN). Signal transducer and activator of transcription (STAT) 3 signaling is activated in podocytes of patients with HIVAN and drives the dedifferentiation and proliferation of podocytes in culture. We confirm here that deletion of podocyte STAT3 is sufficient to mitigate the glomerular as well as tubulointerstitial findings of HIVAN. Methods:To demonstrate the functional role of podocyte STAT3 in the pathogenesis of HIVAN we compared the development of HIVAN in Tg26 HIV-transgenic mice with and without deletion of STAT3 in the podocyte. Results:Tg26 mice with podocyte-specific STAT3 deletion developed significantly less weight loss, albuminuria, and renal function impairment compared to Tg26 mice without STAT3 deletion. Tg26 mice with podocyte STAT3 deletion also had significantly less glomerular collapse, sclerosis, epithelial cell hyperplasia, podocyte dedifferentiation, and proinflammatory STAT3 target gene expression; and tubulointerstitial changes of HIVAN, including tubular atrophy, degeneration, apoptosis, and lymphocyte infiltration, were also significantly reduced compared to Tg26 mice without STAT3 deletion. Conclusion:Development of glomerular as well as tubulointerstitial injuries in the Tg26 HIVAN model is dependent on podocyte STAT3 expression. Inhibition of STAT3 could be a potential adjunctive therapy for the treatment of HIVAN.

Advanced glycation end product accumulation: a new enemy to target in chronic kidney disease?

Purpose of reviewThe critical role of advanced glycation end products (AGEs) in the progression of chronic diseases and their complications has recently become more apparent. This review summarizes the recent contributions to the field of AGEs in chronic kidney disease (CKD). Recent findingsOver the past 3 decades, AGEs have been implicated in the progression of CKD, and specifically diabetic nephropathy. Although numerous in-vitro and in-vivo studies highlight the detrimental role of AGEs accumulation in tissue injury, few prospective human studies or clinical trials show that inhibiting this process ameliorates disease. Nonetheless, recent studies have focused on the novel mechanisms that contribute to end-organ injury as a result of AGEs accumulation, as well as novel targets of therapy in kidney disease. SummaryAs the prevalence and the incidence of CKD rises in the United States, it is essential to identify therapeutic strategies that either delay the progression of CKD or improve mortality in this population. The focus of this review is on highlighting the recent studies that advance our current understanding of the mechanisms mediating AGEs-induced CKD progression, as well as novel treatment strategies that have the potential to abrogate this disease process. Video abstracthttp://links.lww.com/CONH/A12

Reduced Krüppel-like factor 2 expression may aggravate the endothelial injury of diabetic nephropathy.

Kruppel-like Factor 2 (KLF2), a shear-stress inducible transcription factor, has endoprotective effects. In streptozotocin-induced diabetic rats, we found that glomerular Klf2 expression was reduced in comparison to non-diabetic rats. However, normalization of hyperglycemia by insulin treatment increased Klf2 expression to a level higher than that of non-diabetic rats. Consistent with this, we found that Klf2 expression was suppressed by high glucose but increased by insulin in cultured endothelial cells. To determine the role of KLF2 in streptozotocin-induced diabetic nephropathy, we used endothelial cell-specific Klf2 heterozygous knockout mice and found that diabetic knockout mice developed more kidney/glomerular hypertrophy and proteinuria than diabetic wide type mice. Glomerular expression of Vegfa, Flk1, and angiopoietin 2 increased but expression of Flt1, Tie2, and angiopoietin 1 decreased in diabetic knockout compared to diabetic wide type mice. Glomerular expression of ZO-1, glycocalyx, and eNOS was also decreased in diabetic knockout compared to diabetic wide type mice. These data suggest knockdown of Klf2 expression in the endothelial cells induced more endothelial cell injury. Interestingly, podocyte injury was also more prominent in diabetic knockout compared to diabetic wide type mice, indicating a crosstalk between these two cell types. Thus, KLF2 may play a role in glomerular endothelial cell injury in early diabetic nephropathy.

Krüppel-Like Factor 15 Mediates Glucocorticoid-Induced Restoration of Podocyte Differentiation Markers.

Podocyte injury is the inciting event in primary glomerulopathies, such as minimal change disease and primary FSGS, and glucocorticoids remain the initial and often, the primary treatment of choice for these glomerulopathies. Because inflammation is not readily apparent in these diseases, understanding the direct effects of glucocorticoids on the podocyte, independent of the immunomodulatory effects, may lead to the identification of targets downstream of glucocorticoids that minimize toxicity without compromising efficacy. Several studies showed that treatment with glucocorticoids restores podocyte differentiation markers and normal ultrastructure and improves cell survival in murine podocytes. We previously determined that Krüppel-like factor 15 (KLF15), a kidney-enriched zinc finger transcription factor, is required for restoring podocyte differentiation markers in mice and human podocytes under cell stress. Here, we show that in vitro treatment with dexamethasone induced a rapid increase of KLF15 expression in human and murine podocytes and enhanced the affinity of glucocorticoid receptor binding to the promoter region of KLF15 In three independent proteinuric murine models, podocyte-specific loss of Klf15 abrogated dexamethasone-induced podocyte recovery. Furthermore, knockdown of KLF15 reduced cell survival and destabilized the actin cytoskeleton in differentiated human podocytes. Conversely, overexpression of KLF15 stabilized the actin cytoskeleton under cell stress in human podocytes. Finally, the level of KLF15 expression in the podocytes and glomeruli from human biopsy specimens correlated with glucocorticoid responsiveness in 35 patients with minimal change disease or primary FSGS. Thus, these studies identify the critical role of KLF15 in mediating the salutary effects of glucocorticoids in the podocyte.

The podocyte as a direct target for treatment of glomerular disease

The Centers for Disease Control and Prevention estimates more than 10% of adults in the United States, over 20 million Americans, have chronic kidney disease (CKD). A failure to maintain the glomerular filtration barrier directly contributes to the onset of CKD. The visceral epithelial cells, podocytes, are integral to the maintenance of this renal filtration barrier. Direct podocyte injury contributes to the onset and progression of glomerular diseases such as minimal change disease (MCD), focal segmental glomerular sclerosis (FSGS), diabetic nephropathy, and HIV-associated nephropathy (HIVAN). Since podocytes are terminally differentiated with minimal capacity to self-replicate, they are extremely sensitive to cellular injury. In the past two decades, our understanding of the mechanism(s) by which podocyte injury occurs has greatly expanded. With this newfound knowledge, therapeutic strategies have shifted to identifying targets directed specifically at the podocyte. Although the systemic effects of these agents are important, their direct effect on the podocyte proves to be essential in ameliorating glomerular disease. In this review, we highlight the mechanisms by which these agents directly target the podocyte independent of its systemic effects.