Muralikrishna Gangadharan Komala

Effects of SGLT2 inhibition in human kidney proximal tubular cells--renoprotection in diabetic nephropathy?

Sodium/glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents used to treat patients with diabetes mellitus. SGLT2 inhibitors block reabsorption of filtered glucose by inhibiting SGLT2, the primary glucose transporter in the proximal tubular cell (PTC), leading to glycosuria and lowering of serum glucose. We examined the renoprotective effects of the SGLT2 inhibitor empagliflozin to determine whether blocking glucose entry into the kidney PTCs reduced the inflammatory and fibrotic responses of the cell to high glucose. We used an in vitro model of human PTCs. HK2 cells (human kidney PTC line) were exposed to control 5 mM, high glucose (HG) 30 mM or the profibrotic cytokine transforming growth factor beta (TGFβ1; 0.5 ng/ml) in the presence and absence of empagliflozin for up to 72 h. SGLT1 and 2 expression and various inflammatory/fibrotic markers were assessed. A chromatin immunoprecipitation assay was used to determine the binding of phosphorylated smad3 to the promoter region of the SGLT2 gene. Our data showed that TGFβ1 but not HG increased SGLT2 expression and this occurred via phosphorylated smad3. HG induced expression of Toll-like receptor-4, increased nuclear deoxyribonucleic acid binding for nuclear factor kappa B (NF-κB) and activator protein 1, induced collagen IV expression as well as interleukin-6 secretion all of which were attenuated with empagliflozin. Empagliflozin did not reduce high mobility group box protein 1 induced NF-κB suggesting that its effect is specifically related to a reduction in glycotoxicity. SGLT1 and GLUT2 expression was not significantly altered with HG or empagliflozin. In conclusion, empagliflozin reduces HG induced inflammatory and fibrotic markers by blocking glucose transport and did not induce a compensatory increase in SGLT1/GLUT2 expression. Although HG itself does not regulate SGLT2 expression in our model, TGFβ increases SGLT2 expression through phosphorylated smad3.

Sodium glucose cotransporter 2 and the diabetic kidney.

Purpose of reviewReabsorption of glucose in the proximal tubule occurs predominantly via the sodium glucose cotransporter 2 (SGLT2). There has been intense interest in this transporter as a number of SGLT2 inhibitors have entered clinical development. SGLT2 inhibitors act to lower plasma glucose by promoting glycosuria and this review aims to outline the effect on the diabetic kidney of this hypoglycaemic agent. Recent findingsThis review provides an overview of recent findings in this area: the transcriptional control of SGLT2 expression in human proximal tubular cells implicates a number of cytokines in the alteration of SGLT2 expression; experimental data show that SGLT2 inhibition may correct early detrimental effects of diabetes by reducing proximal tubular sodium and glucose transport, suggesting a possible renoprotective effect independent of the glucose lowering effects of these agents; and the nonglycaemic effects of SGLT2 inhibitors may have an impact on renal outcomes. SummaryThe available clinical evidence shows consistent reduction in glycaemic parameters and some evidence suggests additional effects including weight loss and mild blood pressure reduction. There are some side effects that warrant further investigation and establishing whether SGLT2 inhibition provides a renal benefit relies on future long-term studies with specific renal end-points.

Inhibition of kidney proximal tubular glucose reabsorption does not prevent against diabetic nephropathy in type 1 diabetic eNOS knockout mice.

Background and Objective Sodium glucose cotransporter 2 (SGLT2) is the main luminal glucose transporter in the kidney. SGLT2 inhibition results in glycosuria and improved glycaemic control. Drugs inhibiting this transporter have recently been approved for clinical use and have been suggested to have potential renoprotective benefits by limiting glycotoxicity in the proximal tubule. We aimed to determine the renoprotective benefits of empagliflozin, an SGLT2 inhibitor, independent of its glucose lowering effect. Research Design and Methods We induced diabetes using a low dose streptozotocin protocol in 7–8 week old endothelial nitric oxide (eNOS) synthase knockout mice. We measured fasting blood glucose on a monthly basis, terminal urinary albumin/creatinine ratio. Renal histology was assessed for inflammatory and fibrotic changes. Renal cortical mRNA transcription of inflammatory and profibrotic cytokines, glucose transporters and protein expression of SGLT2 and GLUT1 were determined. Outcomes were compared to diabetic animals receiving the angiotensin receptor blocker telmisartan (current best practice). Results Diabetic mice had high matched blood glucose levels. Empagliflozin did not attenuate diabetes-induced albuminuria, unlike telmisartan. Empagliflozin did not improve glomerulosclerosis, tubular atrophy, tubulointerstitial inflammation or fibrosis, while telmisartan attenuated these. Empagliflozin did not modify tubular toll-like receptor-2 expression in diabetic mice. Empagliflozin did not reduce the upregulation of macrophage chemoattractant protein-1 (MCP-1), transforming growth factor β1 and fibronectin mRNA observed in the diabetic animals, while telmisartan decreased transcription of MCP-1 and fibronectin. Empagliflozin increased GLUT1 mRNA expression and telmisartan increased SGLT2 mRNA expression in comparison to untreated diabetic mice. However no significant difference was found in protein expression of GLUT1 or SGLT2 among the different groups. Conclusion Hence SGLT2 inhibition does not have renoprotective benefits independent of glucose lowering.

Saxagliptin reduces renal tubulointerstitial inflammation, hypertrophy and fibrosis in diabetes

In addition to lowering blood glucose in patients with type 2 diabetes mellitus, dipeptidyl peptidase 4 (DPP4) inhibitors have been shown to be antifibrotic and anti‐inflammatory. We have previously shown that DPP4 inhibition in human kidney proximal tubular cells exposed to high glucose reduced fibrotic and inflammatory markers. Hence, we wanted to demonstrate renoprotection in an in vivo model.

Linagliptin Limits High Glucose Induced Conversion of Latent to Active TGFß through Interaction with CIM6PR and Limits Renal Tubulointerstitial Fibronectin.

Background In addition to lowering blood glucose in patients with type 2 diabetes mellitus, dipeptidyl peptidase 4 (DPP4) inhibitors have been shown to be antifibrotic. We have previously shown that cation independent mannose-6-phosphate receptor (CIM6PR) facilitates the conversion of latent to active transforming growth factor β1 (GFß1) in renal proximal tubular cells (PTCs) and linagliptin (a DPP4 inhibitor) reduced this conversion with downstream reduction in fibronectin transcription. Objective We wanted to demonstrate that linagliptin reduces high glucose induced interaction between membrane bound DPP4 and CIM6PR in vitro and demonstrate reduction in active TGFß mediated downstream effects in a rodent model of type 1 diabetic nephropathy independent of high glycaemic levels. Materials and Methods We used human kidney 2 (HK2) cells and endothelial nitric oxide synthase knock out mice to explore the mechanism and antifibrotic potential of linagliptin independent of glucose lowering. Using a proximity ligation assay, we show that CIM6PR and DPP4 interaction was increased by high glucose and reduced by linagliptin and excess mannose-6-phosphate (M6P) confirming that linagliptin is operating through an M6P-dependent mechanism. In vivo studies confirmed these TGFß1 pathway related changes and showed reduced fibronectin, phosphorylated smad2 and phosphorylated smad2/3 (pSmad2/3) with an associated trend towards reduction in tubular atrophy, which was independent of glucose lowering. No reduction in albuminuria, glomerulosclerotic index or cortical collagen deposition was observed. Conclusion Linagliptin inhibits activation of TGFß1 through a M6P dependent mechanism. However this in isolation is not sufficient to reverse the multifactorial nature of diabetic nephropathy.

Novel complement factor H gene mutation causing atypical haemolytic uraemic syndrome: early Eculizumab prevents acute dialysis

Abstract We describe the clinical course and response to treatment of atypical haemolytic uraemic syndrome (aHUS) in two sisters presenting to our hospital 6 years apart with a novel complement factor H mutation that has not been described previously in literature and demonstrates the genetic complexity of this ultra-rare disease. The contrast in course and outcome of disease between the two sisters highlights the rapid evolution of management of aHUS, the importance of rapidly establishing a diagnosis, and how minimizing time to eculizumab therapy significantly reduces associated morbidity and mortality.

Effective Removal of κ-Free Light Chains with Hemodialysis Using Fresenius Ultraflux® EMiC®2 Dialyser in a Patient with Myeloma Cast Nephropathy, with Associated Cost Savings.

with cyclophosphamide. For AKI due to probable cast nephropathy, HCO HD with Fresenius Ultraflux ® EMiC ® 2 dialyser, 8 h/ session, 5 days/week was commenced. After 2 weeks of treatments, although κ-FLC Dear Editor, Cast nephropathy is the commonest cause of acute kidney injury (AKI) in patients with multiple myeloma (MM) [1] . There has been a recent interest in extended hemodialysis (HD) with high cut-off (HCO) dialysers for removal of immunoglobulinfree light chains (FLC), combined with effective chemotherapy to improve renal and patient outcomes [2, 3] . The conventionally used Gambro HCO 1100 dialyser is expensive. We report a patient with kappa (κ) FLC MM and AKI with significant reductions in κ-FLC levels using the cheaper Fresenius Ultraflux ® EMiC ® 2 dialyser. A 59-year-old Caucasian lady with a background of recurrent MM after autologous bone marrow transplant, treated with lenalidomide, monthly zoledronic acid infusions and later with bortezomib, presented with another relapse and AKI. She had previously had AKI due to biopsy proven cast nephropathy, acute tubular injury and eosinophilic interstitial nephritis secondary to zoledronic acid that was treated with steroids with near complete recovery of renal functions to a serum creatinine of 113 μmol/l. Bortezomib had to be withheld because of disseminated herpes zoster infection, but 4 months later, κ-FLC levels increased from 105 to 11,367 mg/l, with serum creatinine of 393 μmol/l at presentation. Bortezomib was recommenced along Received: March 14, 2016 Accepted: April 14, 2016 Published online: June 8, 2016

Quiz Page January 2016: A Man With Chronic Lymphocytic Leukemia and Declining Kidney Function

Figure 1. Urine cytology shows a decoy cell (arrow) (original CLINICAL PRESENTATION A 56-year-old man with a history of multinodular goiter after total thyroidectomy, chest wall melanoma treated with curative resection, and hypertension was given a diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). He received 6 cycles of fludarabine, cyclophosphamide, and rituximab. The patient experienced a relapse of CLL 15 months after presentation, and a 6-cycle salvage chemoimmunotherapy protocol with fludarabine and alemtuzumab was planned. After 3 cycles, the patient developed aspergillus meningitis, complicated by posterior circulation stroke and lateral medullary syndrome. He was treated with voriconazole and had near-complete neurologic recovery. Chemo-immunotherapy was resumed after a 3-month interruption, and the remaining 3 cycles were administered. Throughout this period, kidney function remained stable, with serum creatinine levels ranging between 0.7 and 1.1 mg/ dL (corresponding to estimated glomerular filtration rates [GFRs] of 73 to 103 mL/min/1.73 m as calculated by the CKD-EPI [Chronic Kidney Disease Epidemiology Collaboration] equation). After he completed therapy, the patient’s kidney function progressively deteriorated over a 6-month period, with serum creatinine level increasing to 2.9 mg/dL (estimated GFR, 23 mL/min/1.73 m). He remained normotensive, and urinalysis revealed nonselective proteinuria with protein excretion of 0.45 g/d without hematuria or leukocyturia. Renal ultrasound showed normal-sized kidneys with no hydronephrosis. Urine cytology was requested (Fig 1), and a kidney biopsy was performed (Figs 2-4).

Quiz Page January 2016

Figure 1. Urine cytology shows a decoy cell (arrow) (original CLINICAL PRESENTATION A 56-year-old man with a history of multinodular goiter after total thyroidectomy, chest wall melanoma treated with curative resection, and hypertension was given a diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). He received 6 cycles of fludarabine, cyclophosphamide, and rituximab. The patient experienced a relapse of CLL 15 months after presentation, and a 6-cycle salvage chemoimmunotherapy protocol with fludarabine and alemtuzumab was planned. After 3 cycles, the patient developed aspergillus meningitis, complicated by posterior circulation stroke and lateral medullary syndrome. He was treated with voriconazole and had near-complete neurologic recovery. Chemo-immunotherapy was resumed after a 3-month interruption, and the remaining 3 cycles were administered. Throughout this period, kidney function remained stable, with serum creatinine levels ranging between 0.7 and 1.1 mg/ dL (corresponding to estimated glomerular filtration rates [GFRs] of 73 to 103 mL/min/1.73 m as calculated by the CKD-EPI [Chronic Kidney Disease Epidemiology Collaboration] equation). After he completed therapy, the patient’s kidney function progressively deteriorated over a 6-month period, with serum creatinine level increasing to 2.9 mg/dL (estimated GFR, 23 mL/min/1.73 m). He remained normotensive, and urinalysis revealed nonselective proteinuria with protein excretion of 0.45 g/d without hematuria or leukocyturia. Renal ultrasound showed normal-sized kidneys with no hydronephrosis. Urine cytology was requested (Fig 1), and a kidney biopsy was performed (Figs 2-4).